Anti-mertk agonistic antibodies and uses thereof

ABSTRACT

The present disclosure provides antibody-drug conjugates comprising (i) antibodies that specifically bind to Mer Tyrosin Kinase (MERTK) (e.g., human MERTK, or both human and mouse MERTK), and (ii) cytotoxic agents conjugated directly to the antibodies or conjugated to the antibodies via linkers, and compositions comprising such antibody-drug conjugates, wherein the antibodies contained in the antibody-drug conjugates agonize MERTK signaling of endothelial cells. The present disclosure also provides methods for treating cancer, by administering an antibody-drug conjugate that comprises (i) an antibody that specifically binds to MERTK and agonizes MERTK signaling of endothelial cells, and (ii) a cytotoxic agent conjugated directly to the antibody or conjugated to the antibody via a linker.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/525,993, filed Jun. 28, 2017, thedisclosure of which is incorporated herein by reference in its entirety.

PARTIES TO A JOINT RESEARCH AGREEMENT

The subject matter of this application was made as a result ofactivities undertaken within the scope of a joint research agreementbetween The Rockefeller University and Rgenix, Inc. (Rgenix, Inc. beingnow known as Inspirna, Inc.).

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Sequence Listing submittedwith this application as a text file entitled “SequenceListing.txt”created on Jun. 16, 2017 and having a size of 29 kilobytes.

1. FIELD

The present disclosure provides antibody-drug conjugates comprising (i)antibodies that specifically bind to Mer Tyrosine Kinase (MERTK) (e.g.,human MERTK, or both human and mouse MERTK), and (ii) cytotoxic agentsconjugated directly to the antibodies or conjugated to the antibodiesvia linkers, and compositions comprising such antibody-drug conjugates,wherein the antibodies contained in the antibody-drug conjugates agonizeMERTK signaling of endothelial cells. The present disclosure alsoprovides methods for treating cancer, by administering an antibody-drugconjugate that comprises (i) an antibody that specifically binds toMERTK and agonizes MERTK signaling of endothelial cells, and (ii) acytotoxic agent conjugated directly to the antibody or conjugated to theantibody via a linker.

2. BACKGROUND

Mer Tyrosine Kinase (MERTK), also referred to as c-mer, MER,Proto-oncogene c-Mer, Receptor Tyrosine Kinase MerTK, Tyrosine-proteinKinase Mer, STK Kinase, RP38, or MGC133349, is a member of the TAMfamily of receptor tyrosine kinases, which also include AXL and TYRO3kinases. MERTK transduces signals from the extracellular space viaactivation by binding of ligands, most notably Gas-6, a soluble protein.Gas-6 binding to MERTK induces autophosphorylation of MERTK on itsintracellular domain, resulting in downstream signal activation(Cummings C T et al., (2013) Clin Cancer Res 19: 5275-5280; Verma A etal., (2011) Mol Cancer Ther 10: 1763-1773).

The MERTK receptor exists in both membrane bound and soluble forms. Theextracellular domain can be cleaved to generate a soluble extracellulardomain, which is hypothesized to act as a decoy receptor to negativelyregulate MERTK receptor activation on cells by reducing the abilityand/or availability of soluble Gas-6 ligand to bind membrane-bound MERTK(Sather S et al., (2007) Blood 109: 1026-1033). As a result, MERTK hasdual roles related to cancer progression, angiogenesis, and metastasis.On the one hand, Gas-6 activation of MERTK on endothelial cells resultsin inhibition of endothelial cell recruitment by cancer cells in aco-culture system. Endothelial recruitment is a key feature of cancercells that allows for tumor angiogenesis, tumor growth, and metastasis.However, on the other hand, MERTK plays an opposite role in cancercells, where its over-expression leads to increased metastasis, likelyby releasing cleaved MERTK to generate soluble MERTK extracellulardomain protein as a decoy receptor. Thus, tumor cells secrete a solubleform of the extracellular MERTK receptor that acts as a decoy receptorto reduce the ability (and/or availability) of soluble Gas-6 ligand toactivate MERTK on endothelial cells, ultimately leading to endothelialrecruitment, angiogenesis, and cancer progression (Png K J et al.,(2012) Nature 481: 190-194).

Historically, there have been efforts to generate inhibitors, but notactivators, of MERTK for the treatment of cancer (e.g., compoundUNC1062, a potent small molecule MERTK inhibitor developed as ananticancer compound), because MERTK was thought to solely function as anoncogene (Liu J et al., (2013) Eur J Med Chem 65: 83-93; Cummings C T etal., (2013) Clin Cancer Res 19: 5275-5280; Verma A et al., (2011) MolCancer Ther 10: 1763-1773). Given the dual role of MERTK in cancer cellsand endothelial cells, treatment with a molecule that generally resultsin MERTK activation (e.g., on both endothelial cells and cancer cells)could result in increased endothelial cell recruitment and metastasis.However, a compound that activates MERTK signaling of endothelial cellsbut not cancer cells would potentially be an attractive therapeutic fortumor angiogenesis and metastasis.

Thus, there is a need for therapeutic options involving antibodies thatspecifically bind to MERTK and agonize MERTK signaling of endothelialcells.

In recent years, antibody-drug conjugates (ADCs) have become one of thefastest growing classes of cancer therapeutics (Beck A et al., (2017)Nat Rev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep35: art:e00225).

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

3. SUMMARY

The invention provides an antibody-drug conjugate comprising: (a) anantibody moiety that is an antibody or antigen-binding fragment thereofthat specifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) and agonizes human MERTK signaling of endothelial cells;(b) one or more drug moieties, each drug moiety being a cytotoxic agent;and (c) optionally a linker; wherein the cytotoxic agent is conjugateddirectly to the antibody moiety or is conjugated to the antibody moietyvia the linker.

Non-limiting exemplary anti-MERTK antibodies or antigen-bindingfragments thereof that can be the antibody moiety of the antibody-drugconjugates of the invention are described below.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof described herein specifically recognizes theextracellular domain of human MERTK. In another specific embodiment, ananti-MERTK antibody or antigen-binding fragment thereof described hereinspecifically recognizes the extracellular domain of mouse MERTK. Inanother particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof described herein competes with Gas-6 for binding toMERTK (e.g., human MERTK, or both human and mouse MERTK). In aparticular embodiment, an anti-MERTK antibody or antigen-bindingfragment described herein is monoclonal. In another particularembodiment, an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein is an immunoglobulin comprising two identical lightchains and two identical heavy chains.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 1) (a) a VH CDR1 of NYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 2) (a) a VH CDR1 of GYTFTNY; and/or (SEQ ID NO: 7)(b) a VH CDR2 of; and/or (SEQ ID NO: 12) (c) a VH CDR3 of STVVSRYFD.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 8)(b) a VH CDR2 of WINTYTGEPT; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 4) (a) a VH CDR1 of TNYGMN; and/or (SEQ ID NO: 9)(b) a VH CDR2 of WMGWINTYTGEPT; and/or (SEQ ID NO: 13)(c) a VH CDR3 of ARKSTVVSRYFD.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 5) (a) a VH CDR1 of GYTFTNYG; and/or (SEQ ID NO: 10)(b) a VH CDR2 of INTYTGEP; and/or (SEQ ID NO: 14)(c) a VH CDR3 of ARKSTVVSRYFDV.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(SEQ ID NO: 15) (a) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(b) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(c) a VL CDR3 of QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 16) SQDVGDA; and/or (b) a VL CDR2 of(SEQ ID NO: 20) WAS; and/or (c) a VL CDR3 of (SEQ ID NO: 23) YRSYPL.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 17) GDAVTWC; and/or (b) a VL CDR2 of(SEQ ID NO: 21) LLIYWASTRH; and/or (c) a VL CDR3 of (SEQ ID NO: 24)QQYRSYPL.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 18) QDVGDA; and/or (b) a VL CDR2 of(SEQ ID NO: 20) WAS; and/or  (c) a VL CDR3 of (SEQ ID NO: 22) QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 25) DYSMH; and/or (b) a VH CDR2 of(SEQ ID NO: 30) WINTDTGEPTYADDFKG; and/or  (c) a VH CDR3 of(SEQ ID NO: 35) WFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 26) NYTFTDY; and/or (b) a VH CDR2 of(SEQ ID NO: 31) TDTG; and/or (c) a VH CDR3 of (SEQ ID NO: 36) FGAMD.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 27) NYTFTDYSMH; and/or (b) a VH CDR2 of(SEQ ID NO: 32) WINTDTGEPT; and/or (c) a VH CDR3 of (SEQ ID NO: 35)WFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 28) TDYSMH; and/or (b) a VH CDR2 of(SEQ ID NO: 33) WVGWINTDTGEPT; and/or (c) a VH CDR3 of (SEQ ID NO: 37)ARWFGAMD.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 29) NYTFTDYS; and/or (b) a VH CDR2 of(SEQ ID NO: 34) INTDTGEP; and/or (c) a VH CDR3 of (SEQ ID NO: 38)ARWFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) a VH CDR1 of (SEQ ID NO: 27) NYTFTDYSMH; and/or (b) a VH CDR2 of(SEQ ID NO: 30) WINTDTGEPTYADDFKG; and/or (c) a VH CDR3 of(SEQ ID NO: 35) WFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 39) KASQDVTNVVA; and/or (b) a VL CDR2 of(SEQ ID NO: 43) SASYRYT; and/or (c) a VL CDR3 of (SEQ ID NO: 46)QQYYRTPRT.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 40) SQDVTNV; and/or (b) a VL CDR2 of(SEQ ID NO: 44) SAS; and/or (c) a VL CDR3 of (SEQ ID NO: 47) YYRTPR.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 41) TNVVAWY; and/or (b) a VL CDR2 of(SEQ ID NO: 45) LLIYSASYRY; and/or (c) a VL CDR3 of (SEQ ID NO: 48)QQYYRTPR.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) a VL CDR1 of (SEQ ID NO: 42) QDVTNV; and/or (b) a VL CDR2 of(SEQ ID NO: 44) SAS; and/or (c) a VL CDR3 of (SEQ ID NO: 46) QQYYRTPRT.

In specific embodiments, the anti-MERTK antibody or antigen-bindingfragment thereof comprises one, two, or all three of the VH CDRs above.In certain embodiments, the antibody or antigen-binding fragment thereofcomprises a VH CDR1 in Table 1 or Table 3. In some embodiments, theanti-MERTK antibody or antigen-binding fragment thereof comprises a VHCDR2 in Table 1 or Table 3. In certain embodiments, the anti-MERTKantibody or antigen-binding fragment thereof comprises a VH CDR3 inTable 1 or Table 3. In certain embodiments, the anti-MERTK antibody orantigen-binding fragment thereof comprises a VH CDR1, a VH CDR2, and aVH CDR3 of the antibody M6 (Table 1). In certain embodiments, theanti-MERTK antibody or antigen-binding fragment thereof comprises a VHCDR1, a VH CDR2, and a VH CDR3 of the antibody M19 (Table 3).

In specific embodiments, the anti-MERTK antibody or antigen-bindingfragment thereof comprises one, two, or all three of the VL CDRs above.In certain embodiments, the anti-MERTK antibody or antigen-bindingfragment thereof comprises a VL CDR1 in Table 2 or Table 4. In someembodiments, the anti-MERTK antibody or antigen-binding fragment thereofcomprises a VL CDR2 in Table 2 or Table 4. In certain embodiments, theanti-MERTK antibody or antigen-binding fragment thereof comprises a VLCDR3 in Table 2 or Table 4. In certain embodiments, the anti-MERTKantibody or antigen-binding fragment thereof comprises a VL CDR1, a VLCDR2, and a VL CDR3 of the antibody M6 (Table 2). In certainembodiments, the anti-MERTK antibody or antigen-binding fragment thereofcomprises a VL CDR1, a VL CDR2, and a VL CDR3 of the antibody M19 (Table4).

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a) a VH CDR1 of (SEQ ID NO: 1) NYGMN; and/or (b) a VH CDR2 of(SEQ ID NO: 6) WINTYTGEPTYADDFKG; and/or (c) a VH CDR3 of(SEQ ID NO: 11) KSTVVSRYFDV; and/or (d) a VL CDR1 of (SEQ ID NO: 15)KASQDVGDAVT; and/or (e) a VL CDR2 of (SEQ ID NO: 19) WASTRHT; and/or(f) a VL CDR3 of (SEQ ID NO: 22) QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a) a VH CDR1 of (SEQ ID NO: 2) GYTFTNY; and/or (b) a VH CDR2 of;(SEQ ID NO: 7) and/or (c) a VH CDR3 of (SEQ ID NO: 12) STVVSRYFD; and/or(d) a VL CDR1 of (SEQ ID NO: 16) SQDVGDA; and/or (e) a VL CDR2 of(SEQ ID NO: 20) WAS; and/or (f) a VL CDR3 of (SEQ ID NO: 23) YRSYPL.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 8)(b) a VH CDR2 of WINTYTGEPT; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and/or (SEQ ID NO: 15)(d) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(e) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 4) (a) a VH CDR1 of TNYGMN; and/or (SEQ ID NO: 9)(b) a VH CDR2 of WMGWINTYTGEPT; and/or (SEQ ID NO: 13)(c) a VH CDR3 of ARKSTVVSRYFD; and/or (SEQ ID NO: 17)(d) a VL CDR1 of GDAVTWC; and/or (SEQ ID NO: 21)(e) a VL CDR2 of LLIYWASTRH; and/or (SEQ ID NO: 24)(f) a VL CDR3 of QQYRSYPL.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 5) (a) a VH CDR1 of GYTFTNYG; and/or (SEQ ID NO: 10)(b) a VH CDR2 of INTYTGEP; and/or (SEQ ID NO: 14)(c) a VH CDR3 of ARKSTVVSRYFDV; and/or (SEQ ID NO: 18)(d) a VL CDR1 of QDVGDA; and/or (SEQ ID NO: 20)(e) a VL CDR2 comprising, of WAS; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and/or (SEQ ID NO: 15)(d) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(e) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 25) (a) a VH CDR1 of DYSMH; and/or (SEQ ID NO: 30)(b) a VH CDR2 of WINTDTGEPTYADDFKG; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY; and/or (SEQ ID NO: 39)(d) a VL CDR1 of KASQDVTNVVA; and/or (SEQ ID NO: 43)(e) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(f) a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 26) (a) a VH CDR1 of NYTFTDY; and/or (SEQ ID NO: 31)(b) a VH CDR2 of TDTG; and/or (SEQ ID NO: 36) (c) a VH CDR3 of FGAMD;and/or (SEQ ID NO: 40) (d) a VL CDR1 of SQDVTNV; and/or (SEQ ID NO: 44)(e) a VL CDR2 of SAS; and/or (SEQ ID NO: 47) (f) a VL CDR3 of YYRTPR.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 32)(b) a VH CDR2 of WINTDTGEPT; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY; and/or (SEQ ID NO: 39)(d) a VL CDR1 of KASQDVTNVVA; and/or (SEQ ID NO: 43)(e) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(f) a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 28) (a) a VH CDR1 of TDYSMH; and/or (SEQ ID NO: 33)(b) a VH CDR2 of WVGWINTDTGEPT; and/or (SEQ ID NO: 37)(c) a VH CDR3 of ARWFGAMD; and/or (SEQ ID NO: 41)(d) a VL CDR1 of TNVVAWY; and/or (SEQ ID NO: 45)(e) a VL CDR2 of LLIYSASYRY; and/or (SEQ ID NO: 48)(f) a VL CDR3 of QQYYRTPR.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 29) (a) a VH CDR1 of NYTFTDYS; and/or (SEQ ID NO: 34)(b) a VH CDR2 of INTDTGEP; and/or (SEQ ID NO: 38)(c) a VH CDR3 of ARWFGAMDY; and/or (SEQ ID NO: 42)(d) a VL CDR1 of QDVTNV; and/or (SEQ ID NO: 44) (e) a VL CDR2 of SAS;and/or (SEQ ID NO: 46) (f) a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 30)(b) a VH CDR2 of WINTDTGEPTYADDFKG; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY; and/or (SEQ ID NO: 39)(d) a VL CDR1 of KASQDVTNVVA; and/or (SEQ ID NO: 43)(e) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(f) a VL CDR3 of QQYYRTPRT.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK) and agonizes MERTK signaling of endothelialcells comprises a heavy chain variable region sequence comprising theamino acid sequence of SEQ ID NO: 49. In another specific embodiment, ananti-MERTK antibody or antigen-binding fragment thereof thatspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells comprises aheavy chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 51.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK) and agonizes MERTK signaling of endothelialcells comprises a light chain variable region sequence comprising theamino acid sequence of SEQ ID NO: 50. In another specific embodiment, ananti-MERTK antibody or antigen-binding fragment thereof thatspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells comprises alight chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 52.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK) and agonizes MERTK signaling of endothelialcells comprises (a) a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 49; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 50. In another specificembodiment, an antibody or fragment thereof that specifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizesMERTK signaling of endothelial cells comprises (a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 52.

In certain embodiments, an antibody described herein, which specificallybinds to MERTK (e.g., human MERTK, or both human and mouse MERTK) andagonizes MERTK signaling of endothelial cells, comprises heavy and/orlight chain constant regions. In some embodiments, the heavy chainconstant region is selected from the group of human immunoglobulinsconsisting of IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. In certainembodiments, the light chain constant region is selected from the groupof human immunoglobulins consisting of IgGκ and IgGλ. In someembodiments, the antibody comprises a constant region having increasedbinding affinity to one or more human Fc gamma receptor(s). In someembodiments, the antibody comprises a constant region having decreasedbinding affinity to one or more human Fc gamma receptor(s).

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof that specifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK) and agonizes MERTK signaling of endothelialcells is an anti-MERTK antibody or antigen-binding fragment thereof thatbinds to the same epitope of MERTK (e.g., human MERTK, or both human andmouse MERTK) as the antibody described herein. In another embodiment, ananti-MERTK antibody or antigen-binding fragment thereof thatspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells is ananti-MERTK antibody or antigen-binding fragment thereof that competeswith an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein for binding to MERTK (e.g., human MERTK, or both humanand mouse MERTK). In another specific embodiment, an anti-MERTK antibodyor antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells is a first anti-MERTK antibody orantigen-binding fragment thereof that competes with an anti-MERTKantibody or an antigen-binding fragment thereof described herein forbinding to MERTK (e.g., human MERTK, or both human and mouse MERTK),wherein the competition is exhibited as reduced binding of the firstanti-MERTK antibody or antigen-binding fragment thereof to MERTK (e.g.,human MERTK, or both human and mouse MERTK) by more than 80% (e.g., 85%,90%, 95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85%to 95%) in the presence of the anti-MERTK antibody or antigen-bindingfragment thereof described herein.

In specific embodiments, an antibody described herein, whichspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells is a humanantibody, humanized antibody, murine antibody or chimeric antibody. Incertain embodiments, an antibody described herein, which specificallybinds to MERTK (e.g., human MERTK, or both human and mouse MERTK) andagonizes MERTK signaling of endothelial cells binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) with a K_(D) in the range ofabout 3 pM to 400 pM. In specific embodiments, an antibody describedherein is isolated. In specific embodiments, an antibody describedherein is a monoclonal antibody.

In another embodiment, described herein are nucleic acid moleculesencoding a heavy chain variable region and/or a light chain variableregion, or a heavy chain and/or a light chain of an anti-MERTK antibodydescribed herein. In a specific embodiment, the nucleic acid moleculeencodes a heavy chain variable region comprising the nucleic acidsequence of SEQ ID NO: 53 or SEQ ID NO: 55. In another specificembodiment, the nucleic acid molecule encodes a light chain variableregion comprising the nucleic acid sequence of SEQ ID NO: 54 or SEQ IDNO: 56. In specific embodiments, the nucleic acid molecule is isolated.

In certain embodiments, described herein is a vector (e.g., an isolatedvector) comprises a polynucleotide encoding a heavy chain variableregion and/or a light chain variable region, or a heavy chain and/or alight chain of an anti-MERTK antibody described herein. In certainembodiments, described herein is a host cell that comprises thepolynucleotide or vector. Examples of host cells include E. coli,Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB/20, NS0, PER-C6,HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cellsin tissue culture. In a specific embodiment, described herein is amethod of producing an anti-MERTK antibody or antigen-binding fragmentthereof that specifically binds to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) and agonizes MERTK signaling of endothelial cellscomprising culturing a host cell so that the polynucleotide is expressedand the antibody is produced.

In another embodiment, provided herein are pharmaceutical compositionscomprising an antibody-drug conjugate described herein, and apharmaceutically acceptable carrier. The pharmaceutical composition canbe used to treat cancer.

In certain embodiments, provided herein is a method of treating cancerin a subject, comprising administering to the subject an effectiveamount of an antibody-drug conjugate described herein. In anotherembodiment, provided herein is a method for treating cancer in a subjectcomprising administering to the subject a pharmaceutical compositiondescribed herein. In certain embodiments, the method of treating cancerresults in inhibition of the migration of endothelial cells, inhibitionof angiogenesis, and/or inhibition of tumor progression.

In certain embodiments, the cancer treated by the methods providedherein is a cancer of the lung, breast, bone, ovary, stomach, pancreas,larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum,cervix, uterus, endometrium, kidney, bladder, prostate or thyroid. Insome embodiments, the cancer treated is a sarcoma, squamous cellcarcinoma, melanoma, glioma, glioblastoma, neuroblastoma or Kaposi'ssarcomas.

In certain embodiments, the cancer treated by the methods providedherein is breast cancer. In a specific embodiment, the cancer treated bythe methods provided herein is triple-negative breast cancer.

In certain embodiments, the method of treating cancer further comprisesadministering to the subject an additional therapeutic agent. Examplesof additional therapeutic agents that can be administered to a subjectin combination with an antibody-drug conjugate described herein or apharmaceutical composition described herein are described in Sections5.6 and 5.7, infra.

In specific embodiments, the additional therapeutic agents administeredto a subject in combination with an antibody-drug conjugate describedherein is an agent used to treat breast cancer, an agent used to treatmelanoma, an immunotherapy, or an angiogenesis inhibitor. In a specificembodiment, the additional therapeutic agent is an agent used to treatbreast cancer that is selected from the group consisting of Tamoxifen,Raloxifene, Paclitaxel (TAXOL®), Cyclophosphamide, Docetaxel,Vinblastine, Fluorouracil, Everolimus, Trastuzumab,Trastuzumab-Emtansine, Pertuzumab, and Lapatinib Ditosylate.

In a specific embodiment, the additional therapeutic agent is an agentused to treat melanoma that is selected from the group consisting of aBRAF inhibitor, a MEK inhibitor, and Dacarbazine.

In a specific embodiment, the additional therapeutic agent is anantibody that is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1inhibitor.

In a specific embodiment, the additional therapeutic agent is anangiogenesis inhibitor that is selected from the group consisting of aVEGF inhibitor, a VEGFR2 inhibitor, Sunitinib, and Sorafenib.

In specific embodiments, the subject treated by the methods describedherein is a human.

In one aspect, provided herein is an antibody-drug conjugate comprising:(a) an antibody moiety that is an antibody or an antigen-bindingfragment thereof that specifically binds to human Mer Tyrosine Kinase(MERTK), wherein the antibody agonizes human MERTK signaling ofendothelial cells; (b) one or more drug moieties, each drug moiety beinga cytotoxic agent; and (c) optionally a linker; wherein the cytotoxicagent is conjugated directly to the antibody moiety or is conjugated tothe antibody moiety via the linker.

In a specific embodiment of the preceding aspect, the antibody orantigen-binding fragment thereof further specifically binds to murineMERTK.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody specifically recognizes the extracellular domain of humanMERTK, and the extracellular domain comprises the amino acid sequence ofSEQ ID NO: 58.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody competes with Gas-6 for binding to human MERTK.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody is a monoclonal antibody.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody is an immunoglobulin comprising two identical light chainsand two identical heavy chains.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH),which comprises a complementarity determining region (CDR) 1 of SEQ IDNO: 1, a CDR2 of SEQ ID NO: 6, and a CDR3 of SEQ ID NO: 11. In onefurther embodiment of such a specific embodiment, each of the lightchains comprises a variable region (VL), which comprises acomplementarity determining region (CDR) 1 of SEQ ID NO: 15, a CDR2 ofSEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 7, anda CDR3 of SEQ ID NO: 12. In one further embodiment of such a specificembodiment, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 20,and a CDR3 of SEQ ID NO: 23.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 3, a CDR2 of SEQ ID NO: 8, anda CDR3 of SEQ ID NO: 11. In one further embodiment of such a specificembodiment, each of the light chains comprises a variable region (VL),which comprises a complementarity determining region (CDR) 1 of SEQ IDNO: 15, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 9, anda CDR3 of SEQ ID NO: 13. In one further embodiment of such a specificembodiment, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 17, a CDR2 of SEQ ID NO: 21,and a CDR3 of SEQ ID NO: 24.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 5, a CDR2 of SEQ ID NO: 10,and a CDR3 of SEQ ID NO: 14. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises CDR1 of SEQ ID NO: 18, a CDR2 of SEQ IDNO: 20, and a CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises CDR1 of SEQ ID NO: 3, a CDR2 of SEQ ID NO: 6, and aCDR3 of SEQ ID NO: 11. In one further embodiment of such a specificembodiment, each of the light chains comprises a variable region (VL),which comprises a complementarity determining region (CDR) 1 of SEQ IDNO: 15, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO: 30,and a CDR3 of SEQ ID NO: 35. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQID NO: 43, and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 26, a CDR2 of SEQ ID NO: 31,and a CDR3 of SEQ ID NO: 36. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises a CDR1 of SEQ ID NO: 40, a CDR2 of SEQID NO: 44, and a CDR3 of SEQ ID NO: 47.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 32,and a CDR3 of SEQ ID NO: 35. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQID NO: 43, and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 33,and a CDR3 of SEQ ID NO: 37. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises a CDR1 of SEQ ID NO: 41, a CDR2 of SEQID NO: 45, and a CDR3 of SEQ ID NO: 48.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 29, a CDR2 of SEQ ID NO: 34,and a CDR3 of SEQ ID NO: 38.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the heavy chains comprises a variable region (VH)and the VH comprises a CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 30,and a CDR3 of SEQ ID NO: 35. In one further embodiment of such aspecific embodiment, each of the light chains comprises a variableregion (VL) and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQID NO: 43, and a CDR3 of SEQ ID NO: 46. In another further embodiment ofsuch a specific embodiment, each of the light chains comprises avariable region (VL) and the VL comprises a CDR1 of SEQ ID NO: 42, aCDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL),which comprises a complementarity determining region (CDR) 1 of SEQ IDNO: 15, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 20,and a CDR3 of SEQ ID NO: 23.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 17, a CDR2 of SEQ ID NO: 21,and a CDR3 of SEQ ID NO: 24.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 20, anda CDR3 of SEQ ID NO: 22.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43,and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 44,and a CDR3 of SEQ ID NO: 47.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 41, a CDR2 of SEQ ID NO: 45,and a CDR3 of SEQ ID NO: 48.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, each of the light chains comprises a variable region (VL)and the VL comprises a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ ID NO: 44,and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 49. In one further embodiment of such a specificembodiment, the light chain comprises a variable region (VL), whichcomprises SEQ ID NO: 50.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the light chain comprises a variable region (VL), whichcomprises SEQ ID NO: 50.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the heavy chain comprises a variable region (VH), whichcomprises SEQ ID NO: 51. In one further embodiment of such a specificembodiment, the light chain comprises a variable region (VL), whichcomprises SEQ ID NO: 52.

In a specific embodiment of the embodiment wherein the antibody is animmunoglobulin comprising two identical light chains and two identicalheavy chains, the light chain comprises a variable region (VL), whichcomprises SEQ ID NO: 52.

In a specific embodiment of any of the preceding embodiments, whereinthe antibody is an immunoglobulin comprising two identical light chainsand two identical heavy chains, the antibody or antigen-binding fragmentthereof comprises a human-derived constant region. In one furtherembodiment of such a specific embodiment, the heavy chain constantregion has an isotype selected from the group consisting of gamma1,gamma2, gamma3, and gamma4.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment thereof is humanized.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody binds human MERTK with a dissociation constant (K_(D)) inthe range of about 3 picomolar (pM) to 400 pM.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody binds murine MERTK with a K_(D) in the range of about 3 pMto 400 pM.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment inhibits the migration ofendothelial cells in vitro in the presence of breast cancer cells,wherein the migration is inhibited by more than 30% as compared toendothelial cells treated with a control antibody.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment promotes the phosphorylation ofMERTK of human vascular endothelial cells in vitro.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment does not promote thephosphorylation of MERTK on cancer cells in vitro.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment inhibits tumor angiogenesis invivo.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment does not inhibit the migrationof glioblastoma multiforme cell line A172 in an in vitro trans-wellmigration assay in the absence of endothelial cells.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment does not decrease theexpression level of MERTK on the glioblastoma multiforme cell line A172.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof that specifically binds to human MERTK,the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a complementaritydetermining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 6,and a VH CDR3 of SEQ ID NO: 11; (b) one or more drug moieties, each drugmoiety being a cytotoxic agent; and (c) optionally a linker; wherein thecytotoxic agent is conjugated directly to the antibody moiety or isconjugated to the antibody moiety via the linker. In a specificembodiment of such an aspect, the antibody or antigen-binding fragmentthereof of further comprises a light chain variable region (VL), whereinthe VL comprises a complementarity determining region (CDR) 1 of SEQ IDNO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, anda CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 2, aCDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 12; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (c) optionally alinker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 16,a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 23.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 3, aCDR2 of SEQ ID NO: 8, and a CDR3 of SEQ ID NO: 11; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (c) optionally alinker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a complementaritydetermining region (CDR) 1 of SEQ ID NO: 15, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 22.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 4, aCDR2 of SEQ ID NO: 9, and a CDR3 of SEQ ID NO: 13; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (c) optionally alinker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 17,a CDR2 of SEQ ID NO: 21, and a CDR3 of SEQ ID NO: 24.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 5, aCDR2 of SEQ ID NO: 10, and a CDR3 of SEQ ID NO: 14; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (c) optionally alinker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises CDR1 of SEQ ID NO: 18, aCDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 22.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises CDR1 of SEQ ID NO: 3, aCDR2 of SEQ ID NO: 6, and a CDR3 of SEQ ID NO: 11; (b) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (c) optionally alinker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a complementaritydetermining region (CDR) 1 of SEQ ID NO: 15, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 22.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 25,a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 39,a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 26,a CDR2 of SEQ ID NO: 31, and a CDR3 of SEQ ID NO: 36; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 40,a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 47.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 27,a CDR2 of SEQ ID NO: 32, and a CDR3 of SEQ ID NO: 35; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 39,a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 28,a CDR2 of SEQ ID NO: 33, and a CDR3 of SEQ ID NO: 37; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 41,a CDR2 of SEQ ID NO: 45, and a CDR3 of SEQ ID NO: 48.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 29,a CDR2 of SEQ ID NO: 34, and a CDR3 of SEQ ID NO: 38; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (a) an antibody moiety that is an antibody orantigen-binding fragment thereof of that specifically binds to humanMERTK, the antibody or antigen-binding fragment comprising a heavy chainvariable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 27,a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35; (b) one or moredrug moieties, each drug moiety being a cytotoxic agent; and (c)optionally a linker; wherein the cytotoxic agent is conjugated directlyto the antibody moiety or is conjugated to the antibody moiety via thelinker. In a specific embodiment of such an aspect, the antibody orantigen-binding fragment thereof of further comprises a light chainvariable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 39,a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46. In anotherspecific embodiment of such an aspect, the antibody or antigen-bindingfragment thereof of further comprises a light chain variable region(VL), wherein the VL comprises a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ IDNO: 44, and a CDR3 of SEQ ID NO: 46.

In a specific embodiment of any of the preceding aspects, the antibodyis an immunoglobulin, and the antigen-binding fragment is a portion ofan immunoglobulin.

In a specific embodiment of any of the preceding aspects, the antibodyor antigen-binding fragment thereof is an immunoglobulin.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment thereof is humanized.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody or antigen-binding fragment thereof comprises ahuman-derived constant region. In one further embodiment of such aspecific embodiment, the antibody or antigen-binding fragment thereof isa humanized immunoglobulin.

In a specific embodiment of any of the preceding aspects/embodiments,wherein the antibody is not a monoclonal antibody or an immunoglobulincomprising two identical light chains and two identical heavy chains,the antibody or antigen-binding fragment thereof is a bispecificantibody.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (A) an antibody moiety that is an immunoglobulin thatspecifically binds to human MERTK, the immunoglobulin comprising (i) aheavy chain variable region that comprises the amino acid sequence ofSEQ ID NO: 51, and (ii) a light chain variable region that comprises theamino acid sequence of SEQ ID NO: 52; (B) one or more drug moieties,each drug moiety being a cytotoxic agent; and (C) optionally a linker;wherein the cytotoxic agent is conjugated directly to the antibodymoiety or is conjugated to the antibody moiety via the linker.

In another aspect, provided herein is an antibody-drug conjugatecomprising: (A) an antibody moiety that is an immunoglobulin thatspecifically binds to human MERTK, the immunoglobulin comprising (i) aheavy chain variable region that comprises the amino acid sequence ofSEQ ID NO: 49, and (ii) a light chain variable region that comprises theamino acid sequence of SEQ ID NO: 50; (B) one or more drug moieties,each drug moiety being a cytotoxic agent; and (C) optionally a linker;wherein the cytotoxic agent is conjugated directly to the antibodymoiety or is conjugated to the antibody moiety via the linker.

In another aspect, provided herein is an antibody drug conjugatecomprising:

(I) an antibody moiety that is an immunoglobulin that specifically bindsto human MERTK, the immunoglobulin comprising:

-   -   (A) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 51, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 52;        or    -   (B) (i) a heavy chain variable region that comprises the amino        acid sequence of SEQ ID NO: 49, and (ii) a light chain variable        region that comprises the amino acid sequence of SEQ ID NO: 50;

(II) one or more drug moieties, each drug moiety being a cytotoxicagent; and

(III) optionally a linker;

wherein the cytotoxic agent is conjugated directly to the antibodymoiety or is conjugated to the antibody moiety via the linker.

In another aspect, provided herein is an antibody-drug conjugatecomprising:

(I) an antibody moiety that is an antibody which competes for binding toMERTK with a reference antibody selected from the group consisting of:

-   -   (A) a first immunoglobulin comprising (i) a heavy chain variable        region that comprises the amino acid sequence of SEQ ID NO: 51,        and (ii) a light chain variable region that comprises the amino        acid sequence of SEQ ID NO: 52; and    -   (B) a second immunoglobulin comprising (i) a heavy chain        variable region that comprises the amino acid sequence of SEQ ID        NO: 49, and (ii) a light chain variable region that comprises        the amino acid sequence of SEQ ID NO: 50;

(II) one or more drug moieties, each drug moiety being a cytotoxicagent; and

(III) optionally a linker;

wherein the cytotoxic agent is conjugated directly to the antibodymoiety or is conjugated to the antibody moiety via the linker.

In a specific embodiment of any of the preceding aspects/embodiments,the molar ratio of the antibody moiety to the drug moiety is between 1:1and 1:8.

In a specific embodiment of any of the preceding aspects/embodiments,the molar ratio of the antibody moiety to the drug moiety is between 1:3and 1:5.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody-drug conjugate comprises the linker and the linker is acleavable linker.

In a specific embodiment of any of the preceding aspects/embodiments,the antibody-drug conjugate comprises the linker and the linker is anon-cleavable linker.

In a specific embodiment of any of the preceding aspects/embodiments,the cytotoxic agent is a small molecule, a nucleotide, a peptide, or anon-antibody protein. In one further embodiment of such a specificembodiment, the cytotoxic agent is a small molecule.

In a specific embodiment of any of the preceding aspects/embodiments,the cytotoxic agent is an auristatin, a maytansinoid, apyrrolobenzodiazepine, an indolinobenzodiazepine, a calicheamicin, acamptothecin analogue, a duocarmycin, a tubulin inhibitor, a tubulysinor tubulysin analogue, amberstatin269, doxorubicin, an antibiotic, ananthracycline, a microtubule inhibitor, a spliceostatin, or athailanstatin. In one further embodiment of such a specific embodiment,the cytotoxic agent is monomethyl auristatin E (MMAE) or monomethylauristatin F (MMAF). In another further embodiment of such a specificembodiment, the cytotoxic agent is DM1 or DM4.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described in any of the precedingaspects/embodiments wherein the linker is not present, the methodcomprising: (a) conjugating the cytotoxic agent directly to the antibodymoiety to produce the antibody-drug conjugate; and (b) purifying theantibody-drug conjugate.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described in any of the precedingaspects/embodiments wherein the antibody-drug conjugate comprises thelinker, the method comprising the following steps in the order stated:(a) conjugating the linker directly to the antibody moiety to produce alinker-antibody moiety; (b) conjugating the linker of thelinker-antibody moiety directly to the cytotoxic agent to produce theantibody-drug conjugate; and (c) purifying the antibody-drug conjugate.

In another aspect, provided herein is a method of producing anantibody-drug conjugate described in any of the precedingaspects/embodiments wherein the antibody-drug conjugate comprises thelinker, the method comprising the following steps in the order stated:(a) conjugating the linker directly to the cytotoxic agent to produce alinker-cytotoxic agent moiety; (b) conjugating the linker of thelinker-cytotoxic agent moiety directly to the antibody moiety to producethe antibody-drug conjugate; and (c) purifying the antibody-drugconjugate after (b).

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the antibody-drugconjugate described in any of the preceding aspects/embodiments; and apharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating cancer in asubject in need thereof, comprising administering to the subject thepharmaceutical composition described in the preceding aspect.

In a specific embodiment of the preceding aspect of a method of treatingcancer, the cancer is a cancer of the lung, breast, bone, ovary,stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliarytract, colon, rectum, cervix, uterus, endometrium, kidney, bladder,prostate or thyroid. In a certain embodiment of such a specificembodiment, the cancer is breast cancer. In a further embodiment of sucha certain embodiment, the cancer is triple-negative breast cancer.

In a specific embodiment of the preceding aspect of a method of treatingcancer, the cancer is a sarcoma, squamous cell carcinoma, melanoma,glioma, glioblastoma, neuroblastoma or Kaposi's sarcomas.

In a specific embodiment of any of the preceding aspects/embodiments ofa method of treating cancer, the method further comprises administeringto the subject an additional therapeutic agent.

In a specific embodiment of the embodiment wherein the method furthercomprises administering to the subject an additional therapeutic agent,the additional therapeutic agent is for treating the cancer.

In a specific embodiment of the embodiment wherein the additionaltherapeutic agent is for treating the cancer, the additional therapeuticagent is an agent used to treat breast cancer, an agent used to treatmelanoma, an immunotherapy, or an angiogenesis inhibitor. In one furtherembodiment of such a specific embodiment, the additional therapeuticagent is an agent used to treat breast cancer that is selected from thegroup consisting of Tamoxifen, Raloxifene, Paclitaxel, Cyclophosphamide,Docetaxel, Vinblastine, Fluorouracil, Everolimus, Trastuzumab,Trastuzumab-Emtansine, Pertuzumab, and Lapatinib Ditosylate. In anotherfurther embodiment of such a specific embodiment, the additionaltherapeutic agent is an agent used to treat melanoma that is selectedfrom the group consisting of a BRAF inhibitor, a MEK inhibitor, andDacarbazine. In another further embodiment of such a specificembodiment, the additional therapeutic agent is an antibody that is aCTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In anotherfurther embodiment of such a specific embodiment, the additionaltherapeutic agent is an angiogenesis inhibitor that is selected from thegroup consisting of a VEGF inhibitor, a VEGFR2 inhibitor, Sunitinib, andSorafenib.

In a specific embodiment of any of the preceding aspects/embodiments ofa method of treating cancer, the subject is a human.

4. BRIEF DESCRIPTIONS OF FIGURES

FIGS. 1A and 1B: MERTK sequences. A) A schematic of the recombinantMERTK peptide used to immunize mice for the production of antibodies.The peptide consists of the extracellular domain of MERTK (See FIG. 1B),a short linking peptide (IEGRMD), and a portion of human IgG₁. B) Thesequence of full-length human MERTK (SEQ ID NO: 57) indicating theportion of MERTK used in the recombinant MERTK peptide described in FIG.1A (bold and underlined sequence; SEQ ID NO: 58).

FIG. 2 : MERTK monoclonal antibody screen. A diagram showing data fromantibody capture ELISA assays used to characterize binding properties ofMERTK-binding monoclonal antibodies recovered from single hybridomaclones. Antibody clones are arbitrarily designated M1 through M20 in thefirst column. Several of the MERTK binding monoclonal antibodiesdemonstrated high affinity binding to MERTK, as indicated by large(>3.5) O.D. values in the second column. Some of these antibodies alsoneutralized binding of MERTK to Gas-6, as indicated by a low (<2.5)blocking O.D. value in the third column.

FIG. 3 : MERTK binding antibodies M19 and M6 inhibit endothelialrecruitment. A diagram showing that physiological concentrations ofeither monoclonal antibody M6 or M19 is capable of significantlyinhibiting endothelial recruitment by metastatic breast cancer cells.2.5×10⁴ MDA-MB-231 cells were seeded in quadruplicate. Trans-wellmigration of 5×10⁴ HUVEC cells towards the cancer cells was assessed inthe presence of 200 ng/mL control antibody (IgG) or MERTK bindingantibodies isolated from the screen. Images of cells that migratedthrough the trans-well inserts were obtained and cells counted usingImageJ software. N=4. Monoclonal antibodies that significantly inhibitendothelial cell recruitment (M6 and M19) are labeled in red and greenrespectively. Error bars represent standard error of the mean.

FIGS. 4A, 4B and 4C: MERTK-binding antibody M19 activates MERTK onendothelial cells. A) HUVEC cells were treated with either no FBS+noM19, 10% FBS+no M19, 10% FBS+25 μg/mL of M19, or no FBS+25 μg/mL of M19for 16 hours prior to western blot analysis of activated(phosphorylated) MERTK (P-MERTK) and total levels of MERTK and AKT. Asshown, treatment with M19 antibody increased levels of activated MERTK.B) Quantification of MERTK activation with M19 antibody treatment,calculated as a ratio of the protein expression levels of P-MERTK toMERTK, isolated from M19 treated and untreated HUVEC cells from thewestern blot data from FIG. 4 A. C) HUVEC cells were treated withincreasing concentrations of M19 antibody as indicated for 30 minutesprior to western blot analysis of P-MERTK. Note that increasingactivation of MERTK was seen with increasing doses of M19 treatment.

FIGS. 5A and 5B: MERTK binding antibody M6 activates MERTK onendothelial cells. A) HUVEC cells were treated with either no FBS+no M6,10% FBS+no M6, 10% FBS+25 μg/mL of M6, or no FBS+25 μg/mL of M6 for 16hours prior to western blot analysis of activated (phosphorylated) MERTK(P-MERTK) and total levels of MERTK. As shown, treatment with M6antibody increased levels of activated MERTK. B) Quantification of MERTKactivation with M6 antibody treatment, calculated as a ratio of theprotein expression levels of P-MERTK to MERTK, isolated from M6 treatedand untreated HUVEC cells from the western blot data from FIG. 5A.

FIG. 6 : MERTK binding antibody M19 does not activate MERTK on cancercells. LM2 breast cancer cells were treated with either no FBS+no M19antibody, 10% FBS+no M19 antibody, 10% FBS+25 μg/mL M19 antibody or noFBS+25 μg/mL M19 antibody for 16 hours prior to western blot analysis ofactivated MERTK (P-MERTK) and total levels of MERTK. As shown, nodetectable levels of P-MERTK were induced in cancer cells treated withM19 antibody.

FIG. 7 : M19 binds to human MERTK with high affinity. A diagram showingantibody binding kinetics for M19 (Mer-M19) against human MERTK (hMer)using biolayer interferometry. Purified M19 was loaded on an AMQ sensorat a concentration of 1 microgram/mL to test binding against analytes insolution. All analytes were prepared at 5 nM with a 2-fold dilutionseries for a total of 7 concentrations. Kinetic fits were calculatedusing a 1:1 model, with Local fits for each concentration as well as anoverall global fit. The overall global fit calculated binding affinity(K_(D)) for M19 binding to human MERTK was 326 picomolar.

FIG. 8 : M19 binds to murine MERTK with high affinity. A diagram showingantibody binding kinetics for M19 (Mer-M19) against murine MERTK (msMer)using biolayer interferometry. Purified M19 was loaded on an AMQ sensorat a concentration of 1 microgram/mL to test binding against analytes insolution. All analytes were prepared at 5 nM with a 2-fold dilutionseries for a total of 7 concentrations. Kinetic fits were calculatedusing a 1:1 model, with Local fits for each concentration as well as anoverall Global fit. The overall calculated global fit binding affinity(K_(D)) for M19 binding to mouse MERTK was 305 picomolar.

FIGS. 9A and 9B: Treatment with M19 inhibits primary tumor growth andmetastasis of triple-negative breast cancer in vivo. A) Mammary fat padtumor growth of 2000 MDA-MB-231 or 5000 Lm1a1 breast cancer cells inmice treated biweekly with 250 μg of either control antibody (IgG) orM19 antibody. B) Lm1a1 breast cancer cells were injected bilaterally inthe mammary fat pad in mice treated biweekly with 250 μg of eithercontrol antibody (IgG) or M19 antibody. After 98 days the lungs wereremoved, processed for H&E staining, and the number of metastaticnodules were counted. N=4. Error bars represent standard error of themean. P-values were obtained using student's T-test (*p<0.05)

FIG. 10 : Treatment with M19 inhibits angiogenesis in vivo. Mammary fatpad xenograft tumors that were grown for 58 days treated with 250 μg ofeither control antibody (IgG) or M19 antibody twice weekly were doublestained for DAPI and CD31. The vessel density was quantified by using athresholded CD31 signal. N=4. Error bars represent standard error of themean. P-values were obtained using student's T-test (*p<0.05)

FIG. 11 : M6 binds to MERTK with high affinity. M6 binds to human MERTKwith high affinity. A diagram showing antibody binding kinetics for M6(Mer-M6) against human MERTK (hMer) using biolayer interferometry.Purified M6 was loaded on an AMQ sensor at a concentration of 1microgram/mL to test binding against analytes in solution. All analyteswere prepared at 5 nM with a 2-fold dilution series for a total of 7concentrations. Kinetic fits were calculated using a 1:1 model, withLocal fits for each concentration as well as an overall Global fit.Overall calculated binding affinities (K_(D)) for M6 binding to humanMERTK was 4.6 picomolar.

FIG. 12 : Treatment with M6 inhibits primary tumor growth oftriple-negative breast cancer in vivo. Mammary fat pad tumor growth by2000 MDA-MB-231 in mice treated biweekly for 21 days with either 250 μgof control antibody or M6 antibody. N=4. Error bars represent standarderror of the mean.

5. DETAILED DESCRIPTION

Provided herein are antibody-drug conjugates comprising: (a) an antibodymoiety that is an anti-MERTK antibody (e.g., a monoclonal antibody) oran antigen-binding fragment thereof that specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells; (b) one or more drug moieties, each drugmoiety being a cytotoxic agent; and (c) optionally a linker; wherein thecytotoxic agent is conjugated directly to the antibody moiety or isconjugated to the antibody moiety via the linker. The term “conjugated”as used in this disclosure shall mean covalently bound, which can bedirectly or via an intervening covalently bound structure.

In certain embodiments, the antibody-drug conjugate provided herein hasa molar ratio of the antibody moiety to the drug moiety that is between1:1 and 1:20. In specific embodiments, the antibody-drug conjugateprovided herein has a molar ratio of the antibody moiety to the drugmoiety that is between 1:1 and 1:15. In specific embodiments, theantibody-drug conjugate provided herein has a molar ratio of theantibody moiety to the drug moiety that is between 1:1 and 1:12. Inspecific embodiments, the antibody-drug conjugate provided herein has amolar ratio of the antibody moiety to the drug moiety that is between1:1 and 1:8. In preferred embodiments, the antibody-drug conjugateprovided herein has a molar ratio of the antibody moiety to the drugmoiety that is between 1:3 and 1:5. In a specific embodiment, theantibody-drug conjugate provided herein has a molar ratio of theantibody moiety to the drug moiety that is 1:3. In another specificembodiment, the antibody-drug conjugate provided herein has a molarratio of the antibody moiety to the drug moiety that is 1:4. In anotherspecific embodiment, the antibody-drug conjugate provided herein has amolar ratio of the antibody moiety to the drug moiety that is 1:5.

The drug moiety is conjugated to one or more chains of the antibodymoiety. In some embodiments, the drug moiety is conjugated to one chainof the antibody moiety (for example, when the antibody moiety is a scFv,or when the antibody moiety is a multi-chain antibody, such as animmunoglobulin (which is a tetramer), or antigen-binding fragmentthereof). In other embodiments, the drug moiety is conjugated to two ormore chains of the antibody moiety (when the antibody moiety is amulti-chain antibody, such as an immunoglobulin, or antigen-bindingfragment thereof). In a specific embodiment, the drug moiety isconjugated to two identical chains of an immunoglobulin, e.g., the heavychains or the light chains. In other embodiments, the drug moiety isconjugated to all chains of the antibody moiety (when the antibodymoiety is a multi-chain antibody, such as an immunoglobulin orantigen-binding fragment thereof).

In a specific embodiment, the drug moiety is conjugated to one or moresites in the constant region of an antibody. In a particular embodiment,the drug moiety is conjugated to the Fc region of an antibody that is animmunoglobulin.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein specifically recognizes theextracellular domain of MERTK (e.g., human MERTK, or both human andmouse MERTK). In a particular embodiment, an anti-MERTK antibody orantigen-binding fragment thereof described herein does not bind a 185kilodalton (kD) MERTK glycoform expressed in Jurkat cells, a 205 kDMERTK glycoform in the monocytic cell line U937, 135-140 kD MERTKglycoforms expressed in human leukemia cells (e.g. human T-cell acutelymphoblastic leukemia) or 170-190 kD MERTK glycoforms expressed inhuman leukemia cells (e.g. human T-cell acute lymphoblastic leukemia).In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein does not decrease the expression levelof MERTK on cancer cells. In a particular embodiment, an anti-MERTKantibody or antigen-binding fragment thereof described herein does notdecrease the expression level of MERTK on the glioblastoma multiformecell line A172. In specific embodiments, an anti-MERTK antibody orantigen-binding fragment thereof described herein is isolated. In aspecific embodiment, an anti-MERTK antibody or antigen-binding fragmentthereof described herein specifically binds to human MERTK proteincomprising the amino acid sequence of SEQ ID NO: 57. In another specificembodiment, an anti-MERTK antibody or antigen-binding fragment thereofdescribed herein specifically binds to the extracellular region of humanMERTK, comprising the amino acid sequence of SEQ ID NO: 58. In anotherspecific embodiment, an anti-MERTK antibody or antigen-binding fragmentthereof described herein specifically binds to SEQ ID NO: 58. In aspecific embodiment, an anti-MERTK antibody or antigen-binding fragmentthereof described herein comprises two antigen-binding sites that bindto MERTK; in a particular embodiment, the two antigen-binding sites bindto the same epitope on MERTK; in a particular embodiment, thetwo-antigen binding sites comprise identical CDRs.

Antibodies can include, for example, monoclonal antibodies, polyclonalantibodies, recombinantly produced antibodies, monospecific antibodies,multispecific antibodies (including bispecific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer, anantibody heavy chain monomer, an antibody light chain dimer, an antibodyheavy chain dimer, an antibody light chain-antibody heavy chain pair,intrabodies, heteroconjugate antibodies, single domain antibodies,monovalent antibodies, single chain antibodies or single-chain Fvs(scFv), camelized antibodies, affybodies, disulfide-linked Fvs (sdFv),and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Idantibodies). In certain embodiments, antibodies described herein arepolyclonal antibodies. In a preferred embodiment, the antibodiesdescribed herein are monoclonal antibodies. Antibodies can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG₁,IgG₂, IgG₃, IgG₄, IgA₁ or IgA₂), or any subclass (e.g., IgG_(2a) orIgG_(2b)) of immunoglobulin molecule. In certain embodiments, antibodiesdescribed herein are IgG antibodies, or a class or subclass thereof. Ina specific embodiment, the antibody is a humanized monoclonal antibody.In another specific embodiment, the antibody is a human monoclonalantibody, preferably that is an immunoglobulin.

As used herein, the terms “antigen-binding fragment”, “antigen-bindingregion”, and similar terms refer to a portion of an antibody moleculewhich comprises the amino acid residues that confer on the antibodymolecule its specificity for the antigen (e.g., the complementaritydetermining regions (CDR)). The antigen-binding region can be derivedfrom any animal species, such as rodents (e.g., mouse, rat or hamster)and humans. By way of example, antigen-binding fragments include Fabfragments, F(ab′)2 fragments, and antigen binding fragments of any ofthe antibodies described above.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, which differs extensively in sequence amongantibodies and is used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen. In certain embodiments, thevariable region is a human variable region. In certain embodiments, thevariable region comprises rodent or murine CDRs and human frameworkregions (FRs). In particular embodiments, the variable region is aprimate (e.g., non-human primate) variable region. In certainembodiments, the variable region comprises rodent or murine CDRs andprimate (e.g., non-human primate) framework regions (FRs).

CDRs are defined in various ways in the art, including the Kabat,Chothia, AbM, contact, IMGT, and Exemplary definitions. The Kabatdefinition is based on sequence variability and is the most commonlyused definition to predict CDR regions (Kabat, Elvin A. et al.,Sequences of Proteins of Immunological Interest. Bethesda: NationalInstitutes of Health, 1983). The Chothia definition is based on thelocation of the structural loop regions (Chothia et al., (1987) J MolBiol 196: 901-917). The AbM definition, a compromise between the Kabatand Chothia definitions, is an integral suite of programs for antibodystructure modeling produced by the Oxford Molecular Group(bioinforg.uk/abs) (Martin ACR et al., (1989) PNAS 86: 9268-9272). Thecontact definition is based on an analysis of the available complexcrystal structures (bioinforg.uk/abs) (see MacCallum R M et al., (1996)J Mol Biol 5: 732-745). The IMGT definition is from the IMGT (“IMGT®,the international ImMunoGeneTics information System® website imgt.org,founder and director: Marie-Paule Lefranc, Montpellier, France). TheExemplary definition is a combination of AbM and Kabat (Presta et al.,(1997) Cancer Res 57: 4593-4599).

Also described herein are isolated nucleic acids (polynucleotides), suchas complementary DNA (cDNA), encoding such anti-MERTK antibodies, andantigen-binding fragments thereof. In the case where the cytotoxic agentis a peptide or protein, or where the cytotoxic agent and the linker (ifthere is one) are peptides or proteins, nucleic acids encoding theantibody-drug conjugates are also provided. Further described herein arevectors (e.g., expression vectors) and cells (e.g., host cells)comprising nucleic acids (polynucleotides) encoding such anti-MERTKantibodies or antigen-binding fragments thereof or antibody-drugconjugates. Also described herein are methods of making such antibodiesor antibody-drug conjugates.

In other aspects, provided herein are methods of producing theantibody-drug conjugates that comprise (i) the anti-MERTK antibodies orantigen-binding fragments thereof, and (ii) cytotoxic agents conjugateddirectly to the anti-MERTK antibodies or antigen-binding fragments, orconjugated to the anti-MERTK antibodies or antigen-binding fragments vialinkers.

In other aspects, provided herein are methods of treating cancer in asubject comprising administering to the subject an effective amount ofthe antibody-drug conjugate that comprises (i) the anti-MERTK antibodyor antigen-binding fragment thereof, and (ii) cytotoxic agentsconjugated directly to the anti-MERTK antibodies or antigen-bindingfragments, or conjugated to the anti-MERTK antibodies or antigen-bindingfragments via linkers. Related compositions (e.g., pharmaceuticalcompositions) and kits are also provided.

5.1. The Antibody Moiety

Non-limiting exemplary anti-MERTK antibodies or antigen-bindingfragments thereof that can be the antibody moiety of the antibody-drugconjugates provided herein are presented below.

5.1.1. Sequences and Variants

In specific embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein comprises the VH CDR1 of the antibodyin Table 1 or 3 as defined by Kabat, Chothia, AbM, Contact, IMGT, orExemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof described herein comprises the VH CDR2of the antibody in Tables 1 or 3 as defined by Kabat, Chothia, AbM,Contact, IMGT, or Exemplary. In some embodiments, an anti-MERTK antibodyor antigen-binding fragment thereof described herein comprises the VHCDR3 of the antibody in Tables 1 or 3 as defined by Kabat, Chothia, AbM,Contact, IMGT, or Exemplary. In some embodiments, an anti-MERTK antibodyor antigen-binding fragment thereof described herein comprises one, twoor all three of VH CDRs of an antibody in Table 1 or 3 (e.g., the VHCDRs in one row of Table 1, for example, all of the Kabat VH CDRs forantibody M6).

In specific embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein comprises the VL CDR1 of the antibodyin Table 2 or 4 as defined by Kabat, Chothia, AbM, Contact, IMGT, orExemplary. In some embodiments, an anti-MERTK antibody orantigen-binding fragment thereof described herein comprises the VL CDR2of the antibody in Tables 2 or 4 as defined by Kabat, Chothia, AbM,Contact, IMGT, or Exemplary. In some embodiments, an anti-MERTK antibodyor antigen-binding fragment thereof described herein comprises the VLCDR3 of the antibody in Tables 2 or 4 as defined by Kabat, Chothia, AbM,Contact, IMGT, or Exemplary. In some embodiments, an anti-MERTK antibodyor antigen-binding fragment thereof described herein comprises one, twoor all three of VL CDRs of an antibody in Table 2 or 4 (e.g., the VLCDRs in one row of Table 2, for example, all of the Kabat VL CDRs forantibody M6).

TABLE 1 Antibody M6 VH CDR Amino Acid Sequences Definitions VH CDR1VH CDR2 VH CDR3 Kabat NYGMN WINTYTGEPTYADDFKG KSTVVSRYFDV (SEQ ID NO: 1)(SEQ ID NO: 6) (SEQ ID NO: 11) Chothia GYTFTNY TYTG STVVSRYFD(SEQ ID NO: 2) (SEQ ID NO: 7) (SEQ ID NO: 12) AbM GYTFTNYGMN WINTYTGEPTKSTVVSRYFDV (SEQ ID NO: 3) (SEQ ID NO: 8) (SEQ ID NO: 11) Contact TNYGMNWMGWINTYTGEPT ARKSTVVSRYFD (SEQ ID NO: 4) (SEQ ID NO: 9) (SEQ ID NO: 13)IMGT GYTFTNYG INTYTGEP ARKSTVVSRYFDV (SEQ ID NO: 5) (SEQ ID NO: 10)(SEQ ID NO: 14) Exemplary GYTFTNYGMN WINTYTGEPTYADDFKG KSTVVSRYFDV(SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 11)

TABLE 2 Antibody M6 VL CDR Amino Acid Sequences Definitions VL CDR1VL CDR2 VL CDR3 Kabat KASQDVGDAVT WASTRHT QQYRSYPLT (SEQ ID NO: 15)(SEQ ID NO: 19) (SEQ ID NO: 22) Chothia SQDVGDA WAS YRSYPL(SEQ ID NO: 16) (SEQ ID NO: 20) (SEQ ID NO: 23) AbM KASQDVGDAVT WASTRHTQQYRSYPLT (SEQ ID NO: 15) (SEQ ID NO: 19) (SEQ ID NO: 22) ContactGDAVTWC LLIYWASTRH QQYRSYPL (SEQ ID NO: 17) (SEQ ID NO: 21)(SEQ ID NO: 24) IMGT QDVGDA WAS QQYRSYPLT (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) Exemplary KASQDVGDAVT WASTRHT QQYRSYPLT(SEQ ID NO: 15) (SEQ ID NO: 19) (SEQ ID NO: 22)

TABLE 3 Antibody M19 VH CDR Amino Acid Sequences Definitions VH CDR1VH CDR2 VH CDR3 Kabat DYSMH WINTDTGEPTYADDFKG WFGAMDY (SEQ ID NO: 25)(SEQ ID NO: 30) (SEQ ID NO: 35) Chothia NYTFTDY TDTG FGAMD(SEQ ID NO: 26) (SEQ ID NO: 31) (SEQ ID NO: 36) AbM NYTFTDYSMHWINTDTGEPT WFGAMDY (SEQ ID NO: 27) (SEQ ID NO: 32) (SEQ ID NO: 35)Contact TDYSMH WVGWINTDTGEPT ARWFGAMD (SEQ ID NO: 28) (SEQ ID NO: 33)(SEQ ID NO: 37) IMGT NYTFTDYS INTDTGEP ARWFGAMDY (SEQ ID NO: 29)(SEQ ID NO: 34) (SEQ ID NO: 38) Exemplary NYTFTDYSMH WINTDTGEPTYADDFKGWFGAMDY (SEQ ID NO: 27) (SEQ ID NO: 30) (SEQ ID NO: 35)

TABLE 4 Antibody M19 VL CDR Amino Acid Sequences Definitions VL CDR1VL CDR2 VL CDR3 Kabat KASQDVTNVVA SASYRYT QQYYRTPRT (SEQ ID NO: 39)(SEQ ID NO: 43) (SEQ ID NO: 46) Chothia SQDVTNV SAS YYRTPR(SEQ ID NO: 40) (SEQ ID NO: 44) (SEQ ID NO: 47) AbM KASQDVTNVVA SASYRYTQQYYRTPRT (SEQ ID NO: 39) (SEQ ID NO: 43) (SEQ ID NO: 46) ContactTNVVAWY LLIYSASYRY QQYYRTPR (SEQ ID NO: 41) (SEQ ID NO: 45)(SEQ ID NO: 48) IMGT QDVTNV SAS QQYYRTPRT (SEQ ID NO: 42)(SEQ ID NO: 44) (SEQ ID NO: 46) Exemplary KASQDVTNVVA SASYRYT QQYYRTPRT(SEQ ID NO: 39) (SEQ ID NO: 43) (SEQ ID NO: 46)

TABLE 5 Antibody M6 Variable Region Amino Acid Sequences VHQVKLEESGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAYLQINNLKNEDMATYFCARKSTVVSRYFDVWGAGTTVTVSS (SEQ ID NO: 49) VLDIVLTQTHKFMSTSVGDRVSITCKASQDVGDAVTWCQQKPGQPPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVQSEDLADY FCQQYRSYPLTFGAGTKLELKR(SEQ ID NO: 50)

TABLE 6 Antibody M19 Variable Region Amino Acid Sequences VHEVQLEESGPDLKKPGETVKISCKASNYTFTDYSMHWVKQAPGKGLKWVGWINTDTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNED TATYFCARWFGAMDYWGQGTSVTVSS(SEQ ID NO: 51) VL DIVITQSHKFMSTSVGDRVSITCKASQDVTNVVAWYQQKPGQSPKWYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQYY RTPRTFGGGTKLEIKR(SEQ ID NO: 52)

TABLE 7 Antibody M6 Variable Region DNA Sequences VHCAGGTTAAGCTGGAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTACTTGCAGATCAACAACCTCAAAAATGAGGACATGGCCACATATTTCTGTGCAAGAAAAAGTACGGTAGTAAGTAGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 53) VLGGGATATTGTGCTGACACAGACTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTGATGCTGTAACCTGGTGTCAACAGAAACCAGGTCAACCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATTAACAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATCGCAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTG (SEQ ID NO: 54)

Table 8 Antibody M19 Variable Region DNA Sequences VHGAGGTCCAGCTGGAGGAGTCTGGACCTGACCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTAATTATACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGGTGGGCTGGATAAACACTGACACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGCTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTACAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGATGGTTTGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATC TGTCTATTCC(SEQ ID NO: 55) VL GGGATATTGTGATCACACAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGACT  AATGTTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATCGTACTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA ACGG (SEQ ID NO: 56)

TABLE 9  MERTK Protein Sequences Full-Length MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGP HumanFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTG MERTKNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFN (Swiss-Prot CSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDE ID: Q12866.2)VTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFA DDSSEGSEVLM (SEQ ID NO: 57)Extra- REEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPAL cellular MFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVG Doman ofHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLG MERTK UsedAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKM forKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFN ImmunizationLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIP AHGWVDYAPSSTPAPGNA(SEQ ID NO: 58)

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)  (SEQ ID NO: 1) a VH CDR1 of NYGMN; and/or (b) (SEQ ID NO: 6)a VH CDR2 of WINTYTGEPTYADDFKG; and/or   (c) (SEQ ID NO: 11)a VH CDR3 of KSTVVSRYFDV.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)  (SEQ ID NO: 2)  a VH CDR1 of GYTFTNY; and/or (b) (SEQ ID NO: 7)a VH CDR2 of TYTG;  and/or (c) (SEQ ID NO: 12) a VH CDR3 of STVVSRYFD.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)    (SEQ ID NO: 3) a VH CDR1 of GYTFTNYGMN; and/or (b)(SEQ ID NO: 8)  a VH CDR2 of WINTYTGEPT; and/or (c) (SEQ ID NO: 11)a VH CDR3 of KSTVVSRYFDV

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)    (SEQ ID NO: 4) a VH CDR1 of TNYGMN; and/or (b) (SEQ ID NO: 9)a VH CDR2 of WMGWINTYTGEPT; and/or (c) (SEQ ID NO: 13)a VH CDR3 of ARKSTVVSRYFD.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)    (SEQ ID NO: 5) a VH CDR1 of GYTFTNYG; and/or (b) (SEQ ID NO: 10)a VH CDR2 of INTYTGEP; and/or (c) (SEQ ID NO: 14)a VH CDR3 of ARKSTVVSRYFDV.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a)    (SEQ ID NO: 3) a VH CDR1 of GYTFTNYGMN; and/or (b)(SEQ ID NO: 6)  a VH CDR2 of WINTYTGEPTYADDFKG; and/or (c)(SEQ ID NO: 11) a VH CDR3 of KSTVVSRYFDV.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) (SEQ ID NO: 15) a VL CDR1 of KASQDVGDAVT;   and/or (b)(SEQ ID NO: 19) a VL CDR2 of WASTRHT;   and/or (c) (SEQ ID NO: 22)a VL CDR3 of QQYRSYPLT. 

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) (SEQ ID NO: 16) a VL CDR1 of SQDVGDA;   and/or (b) (SEQ ID NO: 20)a VL CDR2 of WAS;   and/or (c) (SEQ ID NO: 23) a VL CDR3 of YRSYPL.

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) (SEQ ID NO: 17)  a VL CDR1 of GDAVTWC;  and/or (b) (SEQ ID NO: 21)a VL CDR2 of LLIYWASTRH;   and/or (c) (SEQ ID NO: 24)a VL CDR3 of QQYRSYPL. 

In another embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(a) (SEQ ID NO: 18)  a VL CDR1 of QDVGDA; and/or (b) (SEQ ID NO: 20)a VL CDR2 comprising, of WAS;  and/or (c) (SEQ ID NO: 22)a VL CDR3 of QQYRSYPLT. 

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(a) (SEQ ID NO: 25) a VH CDR1 of DYSMH;   and/or (b) (SEQ ID NO: 30)a VH CDR2 of WINTDTGEPTYADDFKG;   and/or (c) (SEQ ID NO: 35)a VH CDR3 of WFGAMDY. 

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 26) (a) a VH CDR1 of NYTFTDY; and/or (SEQ ID NO: 31)(b) a VH CDR2 of TDTG; and/or (SEQ ID NO: 36) (c) a VH CDR3 of FGAMD.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 32)(b) a VH CDR2 of WINTDTGEPT; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 28) (a) a VH CDR1 of TDYSMH; and/or (SEQ ID NO: 33)(b) a VH CDR2 of WVGWINTDTGEPT; and/or (SEQ ID NO: 37)(c) a VH CDR3 of ARWFGAMD.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 29) (a) a VH CDR1 of NYTFTDYS; and/or (SEQ ID NO: 34)(b) a VH CDR2 of INTDTGEP; and/or (SEQ ID NO: 38)(c) a VH CDR3 of ARWFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable region(VH) comprising:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 30)(b) a VH CDR2 of WINTDTGEPTYADDFKG; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(SEQ ID NO: 39) (a) a VL CDR1 of KASQDVTNVVA; and/or (SEQ ID NO: 43)(b) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(c) a VL CDR3 of QQYYRTPRT.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(SEQ ID NO: 40) (a) a VL CDR1 of SQDVTNV; and/or (SEQ ID NO: 44)(b) a VL CDR2 of SAS; and/or (SEQ ID NO: 47) (c) a VL CDR3 of YYRTPR.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(SEQ ID NO: 41) (a) a VL CDR1 of TNVVAWY; and/or (SEQ ID NO: 45)(b) a VL CDR2 of LLIYSASYRY; and/or (SEQ ID NO: 48)(c) a VL CDR3 of QQYYRTPR.

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable region(VL) comprising:

(SEQ ID NO: 42) (a) a VL CDR1 of QDVTNV; and/or (SEQ ID NO: 44)(b) a VL CDR2 of SAS; and/or (SEQ ID NO: 46) (c) a VL CDR3 of QQYYRTPRT.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, or all three of the VH CDRs above. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises a VH CDR1 in Table 1 or Table 3. In some embodiments, theantibody or antigen-binding fragment thereof comprises a VH CDR2 inTable 1 or Table 3. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VH CDR3 in Table 1 or Table3. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the antibody M6(Table 1). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VH CDR1, a VH CDR2, and a VH CDR3 of theantibody M19 (Table 3).

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, or all three of the VL CDRs above. Incertain embodiments, the antibody or antigen-binding fragment thereofcomprises a VL CDR1 in Table 2 or Table 4. In some embodiments, theantibody or antigen-binding fragment thereof comprises a VL CDR2 inTable 2 or Table 4. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VL CDR3 in Table 2 or Table4. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 of the antibody M6(Table 2). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 of theantibody M19 (Table 4).

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 1) (a) a VH CDR1 of NYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and/or (SEQ ID NO: 15)(d) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(e) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 2) (a) a VH CDR1 of GYTFTNY; and/or (SEQ ID NO: 7)(b) a VH CDR2 of TYTG; and/or (SEQ ID NO: 12)(c) a VH CDR3 of STVVSRYFD; and/or (SEQ ID NO: 16)(d) a VL CDR1 of SQDVGDA; and/or (SEQ ID NO: 20)(e) a VL CDR2 of WAS; and/or (SEQ ID NO: 23) (f) a VL CDR3 of YRSYPL.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 8)(b) a VH CDR2 of WINTYTGEPT; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and/or (SEQ ID NO: 15)(d) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(e) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 4) (a) a VH CDR1 of TNYGMN; and/or (SEQ ID NO: 9)(b) a VH CDR2 of WMGWINTYTGEPT; and/or (SEQ ID NO: 13)(c) a VH CDR3 of ARKSTVVSRYFD; and/or (SEQ ID NO: 17)(d) a VL CDR1 of GDAVTWC; and/or (SEQ ID NO: 21)(e) a VL CDR2 of LLIYWASTRH; and/or (SEQ ID NO: 24)(f) a VL CDR3 of QQYRSYPL.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 5) (a) a VH CDR1 of GYTFTNYG; and/or (SEQ ID NO: 10)(b) a VH CDR2 of INTYTGEP; and/or (SEQ ID NO: 14)(c) a VH CDR3 of ARKSTVVSRYFDV; and/or (SEQ ID NO: 18)(d) a VL CDR1 of QDVGDA; and/or (SEQ ID NO: 20)(e) a VL CDR2 comprising, of WAS; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and/or (SEQ ID NO: 15)(d) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(e) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(f) a VL CDR3 of QQYRSYPLT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a) (SEQ ID NO: 25) a VH CDR1 of DYSMH; and/or (b)  (SEQ ID NO: 30)a VH CDR2 of WINTDTGEPTYADDFKG; and/or (c)  (SEQ ID NO: 35)a VH CDR3 of WFGAMDY; and/or (d) (SEQ ID NO: 39)a VL CDR1 of KASQDVTNVVA; and/or (e) (SEQ ID NO: 43)a VL CDR2 of SASYRYT; and/or (f) (SEQ ID NO: 46) a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a)  (SEQ ID NO: 26) a VH CDR1 of NYTFTDY; and/or (b) (SEQ ID NO: 31)a VH CDR2 of TDTG; and/or (c) (SEQ ID NO: 36) a VH CDR3 of FGAMD; and/or (d) (SEQ ID NO: 40) a VL CDR1 of SQDVTNV;  and/or (e)(SEQ ID NO: 44) a VL CDR2 of SAS;  and/or (f)   (SEQ ID NO: 47)a VL CDR3 of YYRTPR.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a)  (SEQ ID NO: 27) a VH CDR1 of NYTFTDYSMH;  and/or (b) (SEQ ID NO: 32) a VH CDR2 of WINTDTGEPT;  and/or (c)  (SEQ ID NO: 35)a VH CDR3 of WFGAMDY;  and/or (d)  (SEQ ID NO: 39)a VL CDR1 of KASQDVTNVVA;  and/or (e) (SEQ ID NO: 43)a VL CDR2 of SASYRYT;  and/or (f)  (SEQ ID NO: 46)a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

—a) (SEQ ID NO: 28) a VH CDR1 of TDYSMH;  and/or (b) (SEQ ID NO: 33)a VH CDR2 of WVGWINTDTGEPT;  and/or (c) (SEQ ID NO: 37)a VH CDR3 of ARWFGAMD;  and/or (d) (SEQ ID NO: 41)a VL CDR1 of TNVVAWY;  and/or (e) (SEQ ID NO: 45)a VL CDR2 of LLIYSASYRY;  and/or (f) (SEQ ID NO: 48)a VL CDR3 of QQYYRTPR.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a)  (SEQ ID NO: 29) a VH CDR1 of NYTFTDYS; and/or (b)  (SEQ ID NO: 34)a VH CDR2 of INTDTGEP;  and/or (c) (SEQ ID NO: 38)a VH CDR3 of ARWFGAMDY;  and/or (d) (SEQ ID NO: 42)a VL CDR1 of QDVTNV;  and/or (e)  (SEQ ID NO: 44) a VL CDR2 of SAS; and/or (f)  (SEQ ID NO: 46) a VL CDR3 of QQYYRTPRT.

In a particular embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises:

(a) (SEQ ID NO: 27) a VH CDR1 of NYTFTDYSMH;  and/or (b) (SEQ ID NO: 30)a VH CDR2 of WINTDTGEPTYADDFKG;  and/or (c) (SEQ ID NO: 35)a VH CDR3 of WFGAMDY;  and/or (d) (SEQ ID NO: 39)a VL CDR1 of KASQDVTNVVA;  and/or (e) (SEQ ID NO: 43)a VL CDR2 of SASYRYT;  and/or (f) (SEQ ID NO: 46)a VL CDR3 of QQYYRTPRT.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises one, two, three, four, five or all six of the CDRsabove. In certain embodiments, the antibody or antigen-binding fragmentthereof comprises a VH CDR1 in Table 1 or Table 3. In some embodiments,the antibody or antigen-binding fragment thereof comprises a VH CDR2 inTable 1 or Table 3. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VH CDR3 in Table 1 or Table3. In some embodiments, the antibody or antigen-binding fragment thereofcomprises a VL CDR1 in Table 2 or Table 4. In some embodiments, theantibody or antigen-binding fragment thereof comprises a VL CDR2 inTable 2 or Table 4. In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VL CDR3 in Table 2 or Table4. In some embodiments, the antibody or antigen-binding fragment thereofcomprises a VH CDR1, a VH CDR2, and a VH CDR3 of the antibody M6 (Table1). In certain embodiments, the antibody or antigen-binding fragmentthereof comprises a VH CDR1, a VH CDR2, and a VH CDR3 of the antibodyM19 (Table 3). In certain embodiments, the antibody or antigen-bindingfragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 of theantibody M6 (Table 2). In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VL CDR1, a VL CDR2, and aVL CDR3 of the antibody M19 (Table 4). In certain embodiments, theantibody or antigen-binding fragment thereof comprises a VL CDR1, a VLCDR2, a VL CDR3, a VH CDR1, a VH CDR2, and a VH CDR3 of the antibody M6(Tables 1 and 2). In certain embodiments, the antibody orantigen-binding fragment thereof comprises a VL CDR1, a VL CDR2, a VLCDR3, a VH CDR1, a VH CDR2, and a VH CDR3 of the antibody M19 (Tables 3and 4).

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(a)  (SEQ ID NO: 25) a VH CDR1 of DYSMH;  and/or (b) (SEQ ID NO: 30)a VH CDR2 of WINTDTGEPTYADDFKG;  and/or (c) (SEQ ID NO: 35)a VH CDR3 of WFGAMDY;  and

(ii) the VL comprises:

(a) (SEQ ID NO: 39) a VL CDR1 of KASQDVTNVVA; and/or (b) (SEQ ID NO: 43)a VL CDR2 of SASYRYT; and/or (c) (SEQ ID NO: 46) a VL CDR3 of QQYYRTPRT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(a)  (SEQ ID NO: 26) a VH CDR1 of NYTFTDY;  and/or (b)  (SEQ ID NO: 31)a VH CDR2 of TDTG;  and/or (c)  (SEQ ID NO: 36) a VH CDR3 of FGAMD;  and

(ii) the VL comprises:

(a) (SEQ ID NO: 40) a VL CDR1 of SQDVTNV;  and/or (b) (SEQ ID NO: 44)a VL CDR2 of SAS;  and/or (c) (SEQ ID NO: 47) a VL CDR3 of YYRTPR.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(a) (SEQ ID NO: 1) a VH CDR1 of NYGMN;  and/or (b) (SEQ ID NO: 6)a VH CDR2 of WINTYTGEPTYADDFKG;  and/or (c) (SEQ ID NO: 11)a VH CDR3 of KSTVVSRYFDV;  and

(ii) the VL comprises:

(a) (SEQ ID NO: 15) a VL CDR1 of KASQDVGDAVT;  and/or (b)(SEQ ID NO: 19) a VL CDR2 of WASTRHT;  and/or (c) (SEQ ID NO: 22)a VL CDR3 of QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(a) (SEQ ID NO: 2) a VH CDR1 of GYTFTNY;  and/or (b) (SEQ ID NO: 7)a VH CDR2 of TYTG;  and/or (c) (SEQ ID NO: 12) a VH CDR3 of STVVSRYFD; and

(ii) the VL comprises:

(a) (SEQ ID NO: 16) a VL CDR1 of SQDVGDA;  and/or (b)  (SEQ ID NO: 20)a VL CDR2 of WAS;  and/or (c) (SEQ ID NO: 23) a VL CDR3 of YRSYPL.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(a) (SEQ ID NO: 3) a VH CDR1 of GYTFTNYGMN;  and/or (b) (SEQ ID NO: 8)a VH CDR2 of WINTYTGEPT; and/or (c) (SEQ ID NO: 11)a VH CDR3 of KSTVVSRYFDV;  and

(ii) the VL comprises:

(SEQ ID NO: 15) (a) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19) (b) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(c) a VL CDR3 of QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 4) (a) a VH CDR1 of TNYGMN; and/or (SEQ ID NO: 9)(b) a VH CDR2 of WMGWINTYTGEPT; and/or (SEQ ID NO: 13)(c) a VH CDR3 of ARKSTVVSRYFD; and

(ii) the VL comprises:

(SEQ ID NO: 17) (a) a VL CDR1 of GDAVTWC; and/or (SEQ ID NO: 21)(b) a VL CDR2 of LLIYWASTRH; and/or (SEQ ID NO: 24)(c) a VL CDR3 of QQYRSYPL.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 5) (a) a VH CDR1 of GYTFTNYG; and/or (SEQ ID NO: 10)(b) a VH CDR2 of INTYTGEP; and/or (SEQ ID NO: 14)(c) a VH CDR3 of ARKSTVVSRYFDV; and

(ii) the VL comprises:

(SEQ ID NO: 18) (a) a VL CDR1 of QDVGDA; and/or (SEQ ID NO: 20)(b) a VL CDR2 comprising, of WAS; and/or (SEQ ID NO: 22)(c) a VL CDR3 of QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 3) (a) a VH CDR1 of GYTFTNYGMN; and/or (SEQ ID NO: 6)(b) a VH CDR2 of WINTYTGEPTYADDFKG; and/or (SEQ ID NO: 11)(c) a VH CDR3 of KSTVVSRYFDV; and

(ii) the VL comprises:

(SEQ ID NO: 15) (a) a VL CDR1 of KASQDVGDAVT; and/or (SEQ ID NO: 19)(b) a VL CDR2 of WASTRHT; and/or (SEQ ID NO: 22)(c) a VL CDR3 of QQYRSYPLT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 32)(b) a VH CDR2 of WINTDTGEPT; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY; and

(ii) the VL comprises:

(SEQ ID NO: 39) (a) a VL CDR1 of KASQDVTNVVA and/or; (SEQ ID NO: 43)(b) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(c) a VL CDR3 of QQYYRTPRT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 28) (a) a VH CDR1 of TDYSMH; and/or (SEQ ID NO: 33)(b) a VH CDR2 of WVGWINTDTGEPT; and/or (SEQ ID NO: 37)(c) a VH CDR3 of ARWFGAMD; and

(ii) the VL comprises:

(SEQ ID NO: 41) (a) a VL CDR1 of TNVVAWY; and/or (SEQ ID NO: 45)(b) a VL CDR2 of LLIYSASYRY; and/or (SEQ ID NO: 48)(c) a VL CDR3 of QQYYRTPR.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 29) (a) a VH CDR1 of NYTFTDYS; and/or (SEQ ID NO: 34)(b) a VH CDR2 of INTDTGEP; and/or (SEQ ID NO: 38)(c) a VH CDR3 of ARWFGAMDY; and

(ii) the VL comprises:

(SEQ ID NO: 42) (a) a VL CDR1 of QDVTNV; and/or (SEQ ID NO: 44)(b) a VL CDR2 of SAS; and/or (SEQ ID NO: 46) (c) a VL CDR3 of QQYYRTPRT.

In another embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof, which specifically binds to MERTK (e.g., human MERTK,or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a heavy chain variable region (VH) and alight chain variable region (VL), wherein

(i) the VH comprises:

(SEQ ID NO: 27) (a) a VH CDR1 of NYTFTDYSMH; and/or (SEQ ID NO: 30)(b) a VH CDR2 of WINTDTGEPTYADDFKG; and/or (SEQ ID NO: 35)(c) a VH CDR3 of WFGAMDY; and

(ii) the VL comprises:

(SEQ ID NO: 39) (a) a VL CDR1 of KASQDVTNVVA; and/or (SEQ ID NO: 43)(b) a VL CDR2 of SASYRYT; and/or (SEQ ID NO: 46)(c) a VL CDR3 of QQYYRTPRT.

In specific embodiments, the VH comprises two or all three of the VHCDRs above and/or the VL comprises two or all three of the VL CDRsabove. In certain embodiments, the VH comprises a VH CDR1 of one of theantibodies in Table 1 or Table 3. In some embodiments, the VH comprisesa VH CDR2 of one of the antibodies in Table 1 or Table 3. In certainembodiments, the VH comprises a VH CDR3 of one of the antibodies inTable 1 or Table 3. In some embodiments, the VL comprises a VL CDR1 ofone of the antibodies in Table 2 or Table 4. In some embodiments, the VLcomprises a VL CDR2 of one of the antibodies in Table 2 or Table 4. Incertain embodiments, the VL comprises a VL CDR3 of one of the antibodiesin Table 2 or Table 4. In some embodiments, the VH comprises a VH CDR1,a VH CDR2, and a VH CDR3 of the antibody M6 (Table 1). In certainembodiments, the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 of theantibody M19 (Table 3). In certain embodiments, the VL comprises a VLCDR1, a VL CDR2, and a VL CDR3 of the antibody M6 (Table 2). In certainembodiments, the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 of theVL CDRs of the antibody M19 (Table 4). In some embodiments, ananti-MERTK antibody or antigen-binding fragment thereof comprises a VHand a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3of the antibody M19 (Table 3) and VL comprise a VL CDR1, a VL CDR2, anda VL CDR3 of the VL CDRs of the antibody M19 (Table 4). In someembodiments, an anti-MERTK antibody or antigen-binding fragment thereofcomprises a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2,and a VH CDR3 of the antibody M6 (Table 1) and VL comprise a VL CDR1, aVL CDR2, and a VL CDR3 of the VL CDRs of the antibody M6 (Table 2).

In a specific embodiment, an anti-MERTK antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a heavy chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 49 (Table 5)(e.g., the VH of antibody M6). In another specific embodiment, anantibody described herein, or an antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells, comprises aheavy chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 51 (Table 6) (e.g., the VH of antibody M19).

In another embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises a light chain variable regionsequence comprising the amino acid sequence of SEQ ID NO: 50 (Table 5)(e.g., the VL of antibody M6). In another specific embodiment, anantibody described herein, or an antigen-binding fragment thereof, whichspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) and agonizes MERTK signaling of endothelial cells, comprises alight chain variable region sequence comprising the amino acid sequenceof SEQ ID NO: 52 (Table 6) (e.g., the VL of antibody M19).

In one specific embodiment, an antibody described herein, or anantigen-binding fragment thereof, which specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells, comprises (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 49; and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO: 50 (e.g., the VH and VL of antibody M6). In another specificembodiment, an antibody described herein, or an antigen-binding fragmentthereof, which specifically binds to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) and agonizes MERTK signaling of endothelialcells, comprises (a) a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 50; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 52 (e.g., the VH and VLof antibody M19).

In a preferred embodiment, the antibody is a humanized antibody, or ahumanized antigen-binding fragment thereof, that specifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and comprises:(A) a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 6, a VH CDR3 ofSEQ ID NO: 11, a VL CDR1 of SEQ ID NO: 15, a VL CDR2 of SEQ ID NO: 19,and a VL CDR3 of SEQ ID NO: 22; (B) a VH CDR1 of SEQ ID NO: 2, a VH CDR2of SEQ ID NO: 7, a VH CDR3 of SEQ ID NO: 12, a VL CDR1 of SEQ ID NO: 16,a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 of SEQ ID NO: 23; (C) a VHCDR1 of SEQ ID NO: 3, a VH CDR2 of SEQ ID NO: 8, a VH CDR3 of SEQ ID NO:11, a VL CDR1 of SEQ ID NO: 15, a VL CDR2 of SEQ ID NO: 19, and a VLCDR3 of SEQ ID NO: 22; (D) a VH CDR1 of SEQ ID NO: 4, a VH CDR2 of SEQID NO: 9, a VH CDR3 of SEQ ID NO: 13, a VL CDR1 of SEQ ID NO: 17, a VLCDR2 of SEQ ID NO: 21, and a VL CDR3 of SEQ ID NO: 24; (E) a VH CDR1 ofSEQ ID NO: 5, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 14, aVL CDR1 of SEQ ID NO: 18, a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 ofSEQ ID NO: 22; or (F) a VH CDR1 of SEQ ID NO: 3, a VH CDR2 of SEQ ID NO:6, a VH CDR3 of SEQ ID NO: 11, a VL CDR1 of SEQ ID NO: 15, a VL CDR2 ofSEQ ID NO: 19, and a VL CDR3 of SEQ ID NO: 22.

In certain aspects, an antibody described herein may be described by itsVH domain alone, or its VL domain alone, or by its three VH CDRs alone,or its three VL CDRs alone. See, for example, Rader C et al., (1998)PNAS 95: 8910-8915, which is incorporated herein by reference in itsentirety, describing the humanization of the mouse anti-αvβ3 antibody byidentifying a complementing light chain or heavy chain, respectively,from a human light chain or heavy chain library, resulting in humanizedantibody variants having affinities as high or higher than the affinityof the original antibody. See also, Clackson T et al., (1991) Nature352: 624-628, which is incorporated herein by reference in its entirety,describing methods of producing antibodies that bind a specific antigenby using a specific VH domain (or VL domain) and screening a library forthe complementary variable domains. See also, Kim S J & Hong H J, (2007)J Microbiol 45: 572-577, which is incorporated herein by reference inits entirety, describing methods of producing antibodies that bind aspecific antigen by using a specific VH domain and screening a library(e.g., human VL library) for complementary VL domains; the selected VLdomains in turn could be used to guide selection of additionalcomplementary (e.g., human) VH domains.

In a specific embodiment, the position of one or more CDRs along the VH(e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regionof an antibody described herein may vary by one, two, three, four, five,or six amino acid positions so long as immunospecific binding to MERTK(e.g., human MERTK, or both human and mouse MERTK) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). In anotherembodiment, the length of one or more CDRs along the VH (e.g., CDR1,CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of anantibody described herein may vary (e.g., be shorter or longer) by one,two, three, four, five, six or more amino acids, so long asimmunospecific binding to MERTK (e.g., human MERTK, or both human andmouse MERTK) is maintained (e.g., substantially maintained, for example,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%). In another embodiment, the amino terminus and/or the carboxyterminus of a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VLCDR3 described herein may be extended or shortened by one, two, three,four, five or more amino acids compared to one or more of the CDRsdescribed herein so long as immunospecific binding to MERTK (e.g., humanMERTK, or both human and mouse MERTK) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%). As used herein, the terms“immunospecifically binds,” “immunospecifically recognizes,”“specifically binds,” and “specifically recognizes” are analogous termsin the context of antibodies and refer to antibodies and antigen-bindingfragments thereof that bind to an antigen (e.g., epitope or immunecomplex) via the antigen-binding sites as understood by one skilled inthe art, and does not exclude cross-reactivity of the antibody orantigen-binding fragment with other antigens. Any method known in theart can be used to ascertain whether immunospecific binding to MERTK(e.g., human MERTK, or both human and mouse MERTK) is maintained, forexample, the binding assays and conditions described in Example 4 andExample 7 (Section 6) provided herein, infra.

In specific aspects, described herein is an anti-MERTK antibody orantigen-binding fragment thereof comprising an antibody heavy chainand/or light chain, e.g., a heavy chain alone, a light chain alone, orboth a heavy chain and a light chain. With respect to the light chain,in a specific embodiment, the light chain of an anti-MERTK antibody oran antigen-binding fragment thereof described herein is a kappa lightchain. In another specific embodiment, the light chain of an anti-MERTKantibody or an antigen-binding fragment thereof described herein is alambda light chain. In yet another specific embodiment, the light chainof an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein is a human kappa light chain or a human lambda lightchain.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) comprises alight chain wherein the amino acid sequence of the variable region ofthe light chain comprises a VL CDR1, VL CDR2, and VL CDR3 having theamino acid sequences of antibody M6 or antibody M19 VL CDRs (i.e., thoselisted in Table 2 and Table 4), and wherein the constant region of thelight chain comprises the amino acid sequence of a kappa light chainconstant region. In another particular embodiment, an anti-MERTKantibody or an antigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) comprises a light chain wherein the amino acid sequence ofthe variable region of the light chain comprises the amino acid sequenceof SEQ ID NO: 50 or SEQ ID NO: 52, and wherein the constant region ofthe light chain comprises the amino acid sequence of a kappa light chainconstant region. As used herein, the term “constant region” or “constantdomain” are interchangeable and have its meaning common in the art. Theconstant region is an antibody portion, e.g., a carboxyl terminalportion of a light and/or heavy chain which is not directly involved inbinding of an antibody to antigen but which can exhibit various effectorfunctions, such as interaction with the Fc receptor. The constant regionof an immunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain. In specificembodiments, the antibody or antigen-binding fragment thereof agonizesMERTK signaling.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) comprises a light chain wherein the amino acid sequence ofthe variable region of the light chain comprises a VL CDR1, VL CDR2, andVL CDR3 having the amino acid sequences of antibody M6 or antibody M19VL CDRs (i.e., those listed in Table 2 and Table 4), and wherein theconstant region of the light chain comprises the amino acid sequence ofa lambda light chain constant region. In another particular embodiment,an anti-MERTK antibody or an antigen-binding fragment thereof describedherein, which immunospecifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK) comprises a light chain wherein the aminoacid sequence of the variable region of the light chain comprise theamino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52, and wherein theconstant region of the light chain comprises the amino acid sequence ofa lambda light chain constant region. In specific embodiments, theantibody or antigen-binding fragment thereof agonizes MERTK signaling.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) comprises a light chain wherein the amino acid sequence ofthe variable region of the light chain comprises a VL CDR1, VL CDR2, andVL CDR3 having the amino acid sequences of antibody M6 or antibody M19VL CDRs (i.e., those listed in Table 2 and Table 4), and wherein theconstant region of the light chain comprises the amino acid sequence ofa human kappa or lambda light chain constant region. In a specificembodiment, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which immunospecifically binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) comprises a light chainwherein the amino acid of the variable region of the light chaincomprises the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52, andwherein the constant region of the light chain comprises the amino acidsequence of a human kappa or lambda light chain constant region. Inspecific embodiments, the antibody or antigen-binding fragment thereofagonizes MERTK signaling. Non-limiting examples of human constant regionsequences have been described in the art, e.g., see Kabat E A et al.,(1991).

With respect to the heavy chain, in a specific embodiment, the heavychain of an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ)or mu (μ) heavy chain. In another specific embodiment, the heavy chainof an antibody described can comprise a human alpha (α), delta (δ),epsilon (ε), gamma (γ) or mu (μ) heavy chain.

In a particular embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) comprises aheavy chain wherein the amino acid sequence of the variable region ofthe heavy chain comprises a VH CDR1, VH CDR2, and VH CDR3 having theamino acid sequences of antibody M6 or antibody M19 VH CDRs (i.e., thoselisted in Table 1 and Table 3), and wherein the constant region of theheavy chain comprises the amino acid sequence of a human gamma (γ) heavychain constant region. In another particular embodiment, an anti-MERTKantibody or an antigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) comprises a heavy chain wherein the amino acid sequence ofthe variable region of the heavy chain comprises the amino acid sequenceof SEQ ID NO: 49 or SEQ ID NO: 51, and wherein the constant region ofthe heavy chain comprises the amino acid sequence of a human gamma (γ)heavy chain constant region. In specific embodiments, the antibody orantigen-binding fragment thereof agonizes MERTK signaling.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) comprises aheavy chain wherein the amino acid sequence of the variable region ofthe heavy chain comprises a VH CDR1, VH CDR2, and VH CDR3 having theamino acid sequences of antibody M6 or antibody M19 VH CDRs (i.e., thoselisted in Table 1 and Table 3), and wherein the constant region of theheavy chain comprises the amino acid of a human heavy chain describedherein or known in the art. In a specific embodiment, an anti-MERTKantibody or an antigen-binding fragment thereof described herein, whichspecifically binds to MERTK (e.g., human MERTK, or both human and mouseMERTK) comprises a heavy chain wherein the amino acid sequence of thevariable region of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 49 or SEQ ID NO: 51, and wherein the constant region of theheavy chain comprises the amino acid of a human heavy chain describedherein or known in the art. In specific embodiments, the antibody orantigen-binding fragment thereof agonizes MERTK signaling. Non-limitingexamples of human constant region sequences have been described in theart, e.g., see Kabat E A et al., (1991) supra.

In a specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) comprises aheavy chain variable region (VH) and a light chain variable region (VL)comprising any amino acid sequences described herein, and wherein theconstant regions comprise the amino acid sequences of the constantregions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, ora human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. Inanother specific embodiment, an anti-MERTK antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a VHand a VL comprising any amino acid sequences described herein, andwherein the constant regions comprise the amino acid sequences of theconstant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulinmolecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂), orany subclass (e.g., IgG_(2a) and IgG_(2b)) of immunoglobulin molecule.In a particular embodiment, the constant regions comprise the amino acidsequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA orIgY immunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄,IgA₁ and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In certain embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an anti-MERTKantibody or an antigen-binding fragment thereof described herein toalter one or more functional properties of the antibody.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an anti-MERTKantibody or an antigen-binding fragment thereof described herein (e.g.,CH2 domain (residues 231-340 of human IgG₁) and/or CH3 domain (residues341-447 of human IgG₁) and/or the hinge region, with numbering accordingto the Kabat numbering system (e.g., the EU index in Kabat)) to increaseor decrease the affinity of the antibody or an antigen-binding fragmentthereof for an Fc receptor (e.g., an activated Fc receptor) on thesurface of an effector cell. Mutations in the Fc region of an anti-MERTKantibody or antigen-binding fragment thereof that decrease or increasethe affinity of an antibody for an Fc receptor and techniques forintroducing such mutations into the Fc receptor or fragment thereof areknown to one of skill in the art. Examples of mutations in the Fcreceptor of an anti-MERTK antibody or antigen-binding fragment thereofthat can be made to alter the affinity of the antibody or anantigen-binding fragment thereof for an Fc receptor are described in,e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Pat. No.6,737,056, and International Publication Nos. WO 02/060919; WO 98/23289;and WO 97/34631, which are incorporated herein by reference.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a glycosylated constant region. In some embodiments, theantibody or antigen-binding fragment thereof comprises anon-glycosylated constant region. Antibodies with reduced fucose contenthave been reported to have an increased affinity for Fc receptors, suchas, e.g., FcγRIIIa. Accordingly, in certain embodiments, the antibodiesor antigen-binding fragments thereof described herein have reducedfucose content or no fucose content. Such antibodies or antigen-bindingfragments thereof can be produced using techniques known to one skilledin the art. For example, the antibodies or antigen-binding fragmentsthereof can be expressed in cells deficient or lacking the ability offucosylation. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies orantigen-binding fragments thereof with reduced fucose content.Alternatively, antibodies or antigen-binding fragments with reducedfucose content or no fucose content can be produced by, e.g.: (i)culturing cells under conditions which prevent or reduce fucosylation;(ii) posttranslational removal of fucose (e.g., with a fucosidaseenzyme); (iii) post-translational addition of the desired carbohydrate,e.g., after recombinant expression of a non-glycosylated glycoprotein;or (iv) purification of the glycoprotein so as to select for antibodiesor antigen-binding fragments thereof which are not fucsoylated. See,e.g., Longmore G D & Schachter H (1982) Carbohydr Res 100: 365-92 andImai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84 for methods forproducing antibodies or antigen-binding fragments thereof with no fucosecontent or reduced fucose content.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) and agonizes MERTK signaling of endothelial cells,comprises a heavy chain and/or a light chain, wherein (i) the heavychain comprises (a) a variable region comprising a VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequences of antibody M6 or antibody M19VH CDRs (i.e., those listed in Table 1 and Table 3) and (b) comprises aconstant heavy chain domain comprising the amino acid sequence of theconstant domain of a human IgG heavy chain; and/or (ii) the light chaincomprises (a) a variable region comprising a VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequences of antibody M6 or antibody M19 VLCDRs (i.e., those listed in Table 2 and Table 4) and (b) a constantlight chain domain comprising the amino acid sequence of the constantdomain of a human IgG.

In another particular embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) and agonizes MERTK signaling of endothelial cells,comprises a heavy chain and/or a light chain, wherein (i) the heavychain comprises (a) a variable region comprising the amino acid sequenceof SEQ ID NO: 49 or SEQ ID NO: 51 and (b) a constant heavy chain domaincomprising the amino acid sequence of the constant domain of a humanIgG; and/or (ii) the light chain comprises a variable region comprisingthe amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52 (b) a constantlight chain domain comprising the amino acid sequence of the constantdomain of a human kappa light chain.

The determination of percent identity between two sequences (e.g., aminoacid sequences or nucleic acid sequences) can also be accomplished usinga mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403. BLAST nucleotidesearches can be performed with the NBLAST nucleotide program parametersset, e.g., for score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described herein. BLAST proteinsearches can be performed with the XBLAST program parameters set, e.g.,to score 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule described herein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul S F et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,National Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof, which immunospecifically binds to MERTK (e.g., humanMERTK, or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a VH having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of the VH of SEQ ID NO: 49 or SEQ ID NO: 51. In certainembodiments, an anti-MERTK antibody or antigen-binding fragment thereof,which immunospecifically binds to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) and agonizes MERTK signaling of endothelialcells, comprises a VH domain having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of the VH of SEQ ID NO: 49 or SEQ ID NO: 51, wherein theantibody or antigen-binding fragment comprises CDRs (e.g., VH CDRsand/or VL CDRs) that are identical to the CDRs (e.g., VH CDRs and/or VLCDRs) set forth in Tables 1 to 4.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof, which immunospecifically binds to MERTK (e.g., humanMERTK, or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises a VL having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of the VL of SEQ ID NO: 50 or SEQ ID NO: 52. In certainembodiments, an anti-MERTK antibody or antigen-binding fragment thereof,which immunospecifically binds to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) and agonizes MERTK signaling of endothelialcells, comprises a VL having at least 80%, at least 85%, at least 90%,at least 95%, or at least 98% sequence identity to the amino acidsequence of the VL of SEQ ID NO: 50 or SEQ ID NO: 52, wherein theantibody or antigen-binding fragment comprises CDRs (e.g., VH CDRsand/or VL CDRs) that are identical to the CDRs (e.g., VH CDRs and/or VLCDRs) set forth in Tables 1 to 4.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof, which immunospecifically binds to MERTK (e.g., humanMERTK, or both human and mouse MERTK) and agonizes MERTK signaling ofendothelial cells, comprises: (i) a VH domain having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the VH domain of SEQ ID NO: 49 or SEQ IDNO: 51; and (ii) a VL domain having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of the VL domain of SEQ ID NO: 50 or SEQ ID NO: 52. In certainembodiments, an anti-MERTK antibody or antigen-binding fragment thereof,which immunospecifically binds to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) and agonizes MERTK signaling of endothelialcells, comprises: (i) a VH domain having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of the VH domain of SEQ ID NO: 49 or SEQ ID NO: 51; and(ii) a VL domain having at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% sequence identity to the amino acid sequenceof the VL domain of SEQ ID NO: 50 or SEQ ID NO: 52, wherein the antibodyor antigen-binding fragment comprises CDRs (e.g., VH CDRs and/or VLCDRs) that are identical to the CDRs (e.g., VH CDRs and/or VL CDRs) setforth in Tables 1 to 4.

In another aspect, described herein are antibodies that bind the same oran overlapping epitope of MERTK (e.g., an epitope of human MERTK, orboth human and mouse MERTK) as an antibody described herein (e.g.,antibody M6 or M19). As used herein, an “epitope” is a term in the artand refers to a localized region of an antigen to which an antibody canspecifically bind. An epitope can be, for example, contiguous aminoacids of a polypeptide (linear or contiguous epitope) or an epitope can,for example, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope of anantibody can be determined by, e.g., NMR spectroscopy, X-ray diffractioncrystallography studies, ELISA assays, hydrogen/deuterium exchangecoupled with mass spectrometry (e.g., MALDI mass spectrometry),array-based oligo-peptide scanning assays, and/or mutagenesis mapping(e.g., site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc; see e.g. Meth Enzymol (1985) volumes 114 &115, eds Wyckoff H W et al; U.S. Patent Application No. 2004/0014194),and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies may be accomplished using anymethod known to one of skill in the art. See, e.g., Champe M et al.,(1995) and Cunningham B C & Wells J A (1989) for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques. In addition, antibodies that recognize and bind to the sameor overlapping epitopes of MERTK (e.g., human MERTK, or both human andmouse MERTK) can be identified using routine techniques such as animmunoassay, for example, by showing the ability of one antibody toblock the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competition binding assays also can be usedto determine whether two antibodies have similar binding specificity foran epitope. Competitive binding can be determined in an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as MERTK. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli C et al.,(1983) Methods Enzymol 9: 242-253); solid phase direct biotin-avidin EIA(see Kirkland T N et al., (1986) J Immunol 137: 3614-9); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Press); solid phase direct label RIA using 1-125 label (see MorelG A et al., (1988) Mol Immunol 25(1): 7-15); solid phase directbiotin-avidin EIA (Cheung R C et al., (1990) Virology 176: 546-52); anddirect labeled RIA. (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically, such an assay involves the use of purified antigen(e.g., MERTK such as human MERTK) bound to a solid surface or cellsbearing either of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin. Competitive inhibition can be measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore. A competition binding assay can be configured in a large number ofdifferent formats using either labeled antigen or labeled antibody. In acommon version of this assay, the antigen is immobilized on a 96-wellplate. The ability of unlabeled antibodies to block the binding oflabeled antibodies to the antigen is then measured using radioactive orenzyme labels. For further details see, for example, Wagener C et al.,(1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J ImmunolMethods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879;Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al.,(1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, EdHarlow E & Lane D editors supra, pp. 386-389.

In certain aspects, competition binding assays can be used to determinewhether an antibody is competitively blocked, e.g., in a dose dependentmanner, by another antibody for example, an antibody binds essentiallythe same epitope, or overlapping epitopes, as a reference antibody, whenthe two antibodies recognize identical or sterically overlappingepitopes in competition binding assays such as competition ELISA assays,which can be configured in all number of different formats, using eitherlabeled antigen or labeled antibody. In a particular embodiment, anantibody can be tested in competition binding assays with an antibodydescribed herein (e.g., antibody M6 or M19).

In another aspect, described herein are antibodies that compete (e.g.,in a dose dependent manner) for binding to MERTK (e.g., human MERTK, orboth human and mouse MERTK) with an antibody described herein (e.g., M6or M19), and agonizes MERTK signaling of endothelial cells, asdetermined using assays known to one of skill in the art or describedherein (e.g., ELISA competitive assays or surface plasmon resonance). Inanother aspect, described herein are antibodies that competitivelyinhibit (e.g., in a dose dependent manner) an antibody described herein(e.g., M6 or M19) from binding to MERTK (e.g., human MERTK, or bothhuman and mouse MERTK), as determined using assays known to one of skillin the art or described herein (e.g., ELISA competitive assays, orsuspension array or surface plasmon resonance).

In certain embodiments, described herein is an antibody that competeswith an antibody described herein for binding to MERTK (e.g., humanMERTK, or both human and mouse MERTK) to the same extent that theantibody described herein self-competes for binding to MERTK (e.g.,human MERTK, or both human and mouse MERTK). In certain embodiments,described herein is a first antibody that competes with an antibodydescribed herein for binding to MERTK (e.g., human MERTK, or both humanand mouse MERTK), wherein the competition is exhibited as reducedbinding of the first antibody to MERTK by more than 80% (e.g., 85%, 90%,95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85% to95%).

In specific aspects, described herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to MERTK (e.g.,human MERTK, or both human and mouse MERTK), with an antibody comprisinga VH domain having the amino acid sequence of SEQ ID NO: 49 or SEQ IDNO: 51, and a VL domain having the amino acid sequence of SEQ ID NO: 50or SEQ ID NO: 52.

In specific aspects, described herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to MERTK (e.g.,human MERTK, or both human and mouse MERTK), with an antibody comprising(i) a VH domain comprising a VH CDR1, VH CDR2, and VH CDR3 having theamino acid sequences of the CDRs of an antibody listed in Table 1 orTable 3; and (ii) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3having the amino acid sequences of the VL CDRs of an antibody listed inTable 2 or Table 4.

In a particular embodiment, described herein is an antibody thatcompetes (e.g., in a dose-dependent manner), for specific binding toMERTK (e.g., human MERTK, or both human and mouse MERTK), with anantibody comprising the VH and VL CDRs of antibody M6 (Table 1 and Table2).

In a particular embodiment, described herein is an antibody thatcompetes (e.g., in a dose-dependent manner), for specific binding toMERTK (e.g., human MERTK, or both human and mouse MERTK), with anantibody comprising the VH and VL CDRs of antibody M19 (Table 3 andTable 4).

In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein immunospecificallybinds to the same or an overlapping epitope of antibody comprising (i) aVH domain comprising a VH CDR1, VH CDR2, and VH CDR3 having the aminoacid sequences of the CDRs of antibody listed in Table 1 or Table 3, and(ii) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3 having theamino acid sequences of the CDRs of antibody listed in Table 2 or Table4.

In a specific embodiment, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein immunospecifically binds to the sameor an overlapping epitope of antibody M6, comprising a VH domain havingan amino acid sequence of SEQ ID NO: 49, and a VL domain having an aminoacid sequence of SEQ ID NO: 50. In another specific embodiment, ananti-MERTK antibody or an antigen-binding fragment thereof describedherein immunospecifically binds to the same or an overlapping epitope ofantibody M19, comprising a VH domain having an amino acid sequence ofSEQ ID NO: 51, and a VL domain having an amino acid sequence of SEQ IDNO: 52. Assays known to one of skill in the art or described herein(e.g., X-ray crystallography, ELISA assays, etc.) can be used todetermine if two antibodies bind to the same epitope.

Affinity can be measured and/or expressed in a number of ways known inthe art, including, but not limited to, equilibrium dissociationconstant (K_(D)), and equilibrium association constant (K_(A)). TheK_(D) can be determined by techniques known to one of ordinary skill inthe art, such as biolayer interferometry. In a specific embodiment, theK_(D) is determined as set forth in Example 4 or Example 7 in Section 6,infra.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, an anti-MERTK antibody orantigen-binding fragment thereof that competes with an antibodydescribed herein for binding to MERTK, or an anti-MERTK antibody orantigen-binding fragment thereof that binds to the same or anoverlapping epitope of an antibody described herein, binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) with a K_(D) of lessthan 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM,1 nM, 0.75 nM, 0.5 nM, 0.25 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, or0.005 nM. In some embodiments, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, an anti-MERTKantibody or antigen-binding fragment thereof that competes with anantibody described herein for binding to MERTK, or an anti-MERTKantibody or antigen-binding fragment thereof that binds to the same oran overlapping epitope of an antibody described herein, binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) with a K_(D) of about7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1nM, 0.75 nM, 0.5 nM, 0.25 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, or0.005 nM. In some embodiments, an anti-MERTK antibody or anantigen-binding fragment thereof described herein, an anti-MERTKantibody or antigen-binding fragment thereof that competes with anantibody described herein for binding to MERTK, or an anti-MERTKantibody or antigen-binding fragment thereof that binds to the same oran overlapping epitope of an antibody described herein, binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) with a K_(D) of about3 pM to 400 pM. In a specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) with a K_(D) of about 0.3nM. In another specific embodiment, an anti-MERTK antibody or anantigen-binding fragment thereof described herein binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) with a K_(D) of about 4.6pM. As used herein, the terms “about” when used to modify a numericvalue or numeric range, indicate that deviations of 5% to 10% above and5% to 10% below the value or range remain within the intended meaning ofthe recited value or range.

In certain embodiments, the epitope of an antibody described herein isused as an immunogen to produce antibodies. See, e.g., Section 5.2 infrafor methods for producing antibodies.

In specific embodiments, the anti-MERTK antibody or an antigen-bindingfragment thereof described herein is engineered or modified by a methodknown in the art to facilitate conjugation with the drug moiety(ies), inparticular to facilitate site-specific conjugation with the drugmoiety(ies), for example, by a method described in Beck A et al., (2017)Nat Rev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep35: art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; or OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. Non-limiting exemplary methodsthat can be used (see Beck A et al., (2017) Nat Rev Drug Discov 16:315-337) to engineer or modify an anti-MERTK antibody or anantigen-binding fragment thereof to facilitate conjugation with the drugmoiety(ies) include adding one or more additional cysteines orselenocysteines, unnatural amino acid engineering, adding one or moreamino acid tags recognizable by certain enzymes that can assist with theconjugation, glycan remodeling, adding an amino-terminal serine, andnative cysteine bridging.

5.1.2. Functional Characteristics of the Antibody Moiety

In some aspects, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which immunospecifically binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonizes MERTK signalingof endothelial cells, increases MERTK (e.g., human MERTK, or both humanand mouse MERTK) activity on endothelial cells at least about 2 fold, 3fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold,20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold,or 100 fold. The MERTK activity can be assessed by methods describedherein or known to one of skill in the art, e.g., by measuring theamount of MERTK phosphorylation. In another specific embodiment, theincrease in MERTK activity as measured by an increase in phosphorylationof MERTK is at least 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, or 30 fold, relative to MERTK activity(e.g., human MERTK, or both human and mouse MERTK activity) onendothelial cells without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to MERTK). Inspecific embodiments, the increase in MERTK activity is at least 50%,60%, 70%, 80%, 90%, or 100%. In specific embodiments, an increase inMERTK activity is assessed as described in Example 3, infra.

In certain embodiments, an anti-MERTK antibody or antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizesMERTK signaling of endothelial cells competes with Gas-6 (e.g., humanGas-6) for binding to MERTK. In some embodiments, the antibody orantigen-binding fragment thereof described herein inhibits (e.g.,completely inhibits or only partially inhibits) Gas-6 from binding toMERTK (e.g., human MERTK, or both human and mouse MERTK). In someembodiments, an anti-MERTK antibody or an antigen-binding fragmentthereof described herein, which immunospecifically binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonizes MERTK signalingof endothelial cells inhibits the binding of Gas-6 (e.g., human or mouseGas-6) to MERTK (e.g., human MERTK, or both human and mouse MERTK) onendothelial cells by more than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25%, 20% or 10% as assessed by an assay known to oneof skill in the art or described herein. In a specific embodiment, theassay that is used to assess inhibition of binding of Gas-6 (e.g., humanGas-6) to MERTK (e.g., human MERTK, or both human and mouse MERTK) inthe presence of an anti-MERTK antibody or antigen-binding fragmentthereof described herein is antibody capture ELISA as described inExample 1, infra.

In a specific embodiment, the level of MERTK phosphorylation inendothelial cells is measured by phospho-MERTK-specific Western blottingas described in Example 3, infra.

In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which promotes (i.e., increases) thelevel of MERTK phosphorylation on endothelial cells, does not promote(i.e., increase) MERTK phosphorylation on cancer cells (e.g., breastcancer cells) in vitro. In a specific embodiment, the level of MERTKphosphorylation in cancer cells is measured by phospho-MERTK-specificWestern blotting as described in Example 3, infra.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizesMERTK signaling of endothelial cells, inhibits the migration ofMERTK-expressing endothelial cells in vitro in the presence of cancercells (e.g., breast cancer cells). In some specific embodiments, themigration of the endothelial cells is inhibited by at least 10%, 20%,30%, 35%, 40%, 50%, or 60% as assessed by methods described hereinand/or known to one of skill in the art. In some specific embodiments,the migration of the endothelial cells is inhibited by at least 40%,45%, 50%, 55%, or 60% as assessed by methods described herein and/orknown to one of skill in the art. The extent of migration can beassessed by methods described herein and/or known to one of skill in theart. The inhibition can be relative to the extent of migration of theMERTK-expressing endothelial cells without any antibody or with anunrelated antibody (e.g., an antibody that does not immunospecificallybind to MERTK). In a specific embodiment, the assay that is used toassess endothelial cell migration is a transwell migration assay asdescribed in Example 2, infra.

In particular embodiments, the antibody or antigen-binding fragmentthereof described herein, which immunospecifically binds to MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonizes MERTK signalingof endothelial cells, does not inhibit the migration of glioblastomamultiforme cell line A172 in an in vitro trans-well migration assay inthe absence of endothelial cells.

In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizesMERTK signaling of endothelial cells, inhibits angiogenesis withintumors. In some embodiments, the inhibition of angiogenesis is by atleast 10%, 20%, 30%, 40%, 50%, 55, 60%, 65%, 70%, or 75%. In a specificembodiment, the inhibition of angiogenesis is at least 50%, 55%, 60%,65%, or 70%. Inhibition of angiogenesis can be assessed by methodsdescribed herein and/or known to one of skill in the art. The inhibitioncan be relative to the level of angiogenesis without any antibody orwith an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to MERTK). In a specific embodiment, the assaythat is used to assess angiogenesis in vivo is as described in Example6, infra.

In certain embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein, which immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizesMERTK signaling of endothelial cells, inhibits tumor (e.g., human breastcancer tumor) progression. The inhibition of tumor progression by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. Tumor progression canbe assessed by methods described herein and/or known to one of skill inthe art. The tumor progression can be relative to the cancer statuswithout any antibody or with an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to MERTK). In a specificembodiment, the assay that is used to assess tumor progression is amurine tumor transplantation model as described in Examples 5 and 8,infra.

5.2. Antibody Production

5.2.1. Producing and Screening Antibodies

Also described herein are methods of producing antibodies orantigen-binding fragments thereof that specifically bind to MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonize MERTK signalingof endothelial cells.

The antibodies or antigen-binding fragments thereof described herein canbe produced by any method known in the art for the synthesis ofantibodies, for example, by chemical synthesis or by recombinantexpression techniques. The methods described herein employs, unlessotherwise indicated, conventional techniques in molecular biology,microbiology, genetic analysis, recombinant DNA, organic chemistry,biochemistry, PCR, oligonucleotide synthesis and modification, nucleicacid hybridization, and related fields within the skill of the art.These techniques are described, for example, in the references citedherein and are fully explained in the literature. See, e.g., Maniatis Tet al., (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press;Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y; Ausubel F M etal., Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences that are encoded by DNA sequences that do notnaturally exist within the antibody germline repertoire of an animal ormammal (e.g., human) in vivo. In a specific embodiment, an antibodydescribed herein is made by a method comprising using human MERTK (SEQID NO: 57) or the extracellular domain thereof (SEQ ID NO: 58) as animmunogen.

In a certain aspect, described herein is a method of making ananti-MERTK antibody or antigen-binding fragment thereof whichimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK) and agonizes MERTK signaling of endothelial cellscomprising culturing a cell or host cell described herein. In a certainaspect, described herein is a method of making an anti-MERTK antibody orantigen-binding fragment thereof which immunospecifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells comprising expressing (e.g.,recombinantly expressing) the antibody or antigen-binding fragmentthereof using a cell or host cell described herein (e.g., a cell or ahost cell comprising polynucleotides encoding an antibody describedherein). In a particular embodiment, the cell is an isolated cell. In aparticular embodiment, the exogenous polynucleotides have beenintroduced into the cell. In a particular embodiment, the method furthercomprises the step of purifying the antibody or antigen-binding fragmentthereof obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. Monoclonal antibodiescan be prepared using a wide variety of techniques known in the artincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced recombinantly from host cells exogenouslyexpressing an antibody described herein or a fragment thereof, forexample, a light chain and/or heavy chain of such antibody. Methods forthe preparation of clonal cell lines and of monoclonal antibodiesexpressed thereby are well known in the art (see, for example, Chapter11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F Met al., supra). For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling G J et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y.,1981); and Kohler G & Milstein C (1975) Nature 256: 495. Methods forproducing and screening for specific antibodies using hybridomatechnology are routine and well known in the art. In some embodiments,mice (or other animals, such as rats, monkeys, donkeys, pigs, sheep,hamster, or dogs) can be immunized with an antigen (e.g., MERTK (e.g.,human MERTK, or both human and mouse MERTK)) and once an immune responseis detected, e.g., antibodies specific for the antigen are detected inthe mouse serum, the mouse spleen is harvested and splenocytes isolated.The splenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against MERTK (e.g., humanMERTK, or both human and mouse MERTK). After hybridoma cells areidentified that produce antibodies of the desired specificity, affinity,and/or activity, the clones may be subcloned, grown, and separated fromthe culture medium by standard methods (Goding J W (Ed), MonoclonalAntibodies: Principles and Practice, supra). The binding specificity ofmonoclonal antibodies produced by hybridoma cells is determined bymethods known in the art, for example, immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

In specific embodiments, disclosed herein are monoclonal antibodies thatare produced by a single cell (e.g., hybridoma or host cell producing arecombinant antibody), wherein the antibody immunospecifically binds toMERTK (e.g., human MERTK, or both human and mouse MERTK) as determined,e.g., by ELISA or other antigen-binding or competitive binding assayknown in the art or as described in Example 1 provided herein. Inparticular embodiments, a monoclonal antibody can be a chimeric antibodyor a humanized antibody. In certain embodiments, a monoclonal antibodyis a monovalent antibody or multivalent (e.g., bivalent) antibody. Inparticular embodiments, a monoclonal antibody is a monospecific ormultispecific antibody (e.g., bispecific antibody).

Antibodies described herein include antibody fragments which recognizeMERTK (e.g., human MERTK, or both human and mouse MERTK) and can begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments described herein can be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). A Fab fragment corresponds to one of the two identical armsof an antibody molecule and contains the complete light chain pairedwith the VH and CH1 domains of the heavy chain. A F(ab′)2 fragmentcontains the two antigen-binding arms of an antibody molecule linked bydisulfide bonds in the hinge region.

Further, the antibodies described herein or antigen-binding fragmentsthereof can also be generated using various phage display methods knownin the art. In phage display methods, functional antibody domains aredisplayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of affected tissues). The DNAencoding the VH and VL domains are recombined together with a scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13, and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to a particular antigen can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies described herein include thosedisclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50;Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene187: 9-18; Burton D R & Barbas C F (1994) Advan Immunol 57: 191-280; PCTApplication No. PCT/GB91/001134; International Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO 97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen-binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043.

In one aspect, to generate whole antibodies, PCR primers including VH orVL nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the VH or VLsequences from a template, e.g., scFv clones. Utilizing cloningtechniques known to those of skill in the art, the PCR amplified VHdomains can be cloned into vectors expressing a VH constant region, andthe PCR amplified VL domains can be cloned into vectors expressing a VLconstant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

Further, antibodies that immunospecifically bind to a MERTK antigen can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”an antigen using techniques well known to those skilled in the art.(See, e.g., Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444; andNissinoff A (1991) J Immunol 147(8): 2429-2438).

In particular embodiments, an antibody described herein, which binds tothe same or an overlapping epitope of MERTK (e.g., human MERTK, or bothhuman and mouse MERTK) as a MERTK agonistic antibody described herein,is a human anti-MERTK antibody or antigen-binding fragment thereof. Inparticular embodiments, an antibody described herein, whichcompetitively blocks (e.g., in a dose-dependent manner) any one of theantibodies described herein, (e.g., M6 or M19) from binding to MERTK(e.g., human MERTK, or both human and mouse MERTK), is a humananti-MERTK antibody or antigen-binding fragment thereof. Humanantibodies can be produced using any method known in the art. Forexample, transgenic mice which are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes, can be used. In particular, the human heavy and light chainimmunoglobulin gene complexes can be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region can beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of an antigen (e.g., MERTK). Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see, e.g., Lonberg N& Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g.,International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735;and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capableof producing human antibodies include the Xenomouse™ (Abgenix, Inc; U.S.Pat. Nos. 6,075,181 and 6,150,184), the HuAb-Mouse™ (Mederex, Inc./GenPharm; U.S. Pat. Nos. 5,545,806 and 5,569, 825), the Trans Chromo Mouse™(Kirin) and the KM Mouse™ (Medarex/Kirin).

Human antibodies which specifically bind to MERTK (e.g., human MERTK, orboth human and mouse MERTK) can be made by a variety of methods known inthe art including phage display methods described above using antibodylibraries derived from human immunoglobulin sequences. See also U.S.Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and InternationalPublication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that immunospecifically bind to atarget antigen (e.g., MERTK (e.g., human MERTK, or both human and mouseMERTK)). Such methods are known and are described in the art, see, e.g.,Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al.,(2005) Human Antibodies 14: 27-31.

In specific embodiments, the methods of producing antibodies orantigen-binding fragments thereof that specifically bind to MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonize MERTK signalingof endothelial cells are as described in Example 1, infra.

In specific embodiments, the methods of screening and selectingantibodies or antigen-binding fragments thereof that specifically bindto MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizeMERTK signaling of endothelial cells are as described in Example 1,infra.

Once an anti-MERTK antibody or an antigen-binding fragment thereofdescribed herein has been produced, it can be purified by any methodknown in the art for purification of an immunoglobulin molecule, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an anti-MERTK antibody or an antigen-bindingfragment thereof described herein is isolated or purified. Generally, anisolated antibody is one that is substantially free of other antibodieswith different antigenic specificities than the isolated antibody. Forexample, in a particular embodiment, a preparation of an antibodydescribed herein is substantially free of cellular material and/orchemical precursors. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations ofantibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”) and/or variants of an antibody, for example,different post-translational modified forms of an antibody or otherdifferent versions of an antibody (e.g., antibody fragments). When theantibody is recombinantly produced, it is also generally substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the proteinpreparation. When the antibody is produced by chemical synthesis, it isgenerally substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, or 5%(by dry weight) of chemical precursors or compounds other than theantibody of interest. In a specific embodiment, antibodies describedherein are isolated or purified.

5.2.2. Polynucleotides

Also described herein are polynucleotides comprising a nucleotidesequence encoding an antibody described herein or an antigen-bindingfragment thereof (e.g., a variable light chain region and/or variableheavy chain region) that immunospecifically binds to a MERTK (e.g.,human MERTK, or both human and mouse MERTK) antigen, and vectors, e.g.,vectors comprising such polynucleotides for their efficient expressionin host cells (e.g., E. coli and mammalian cells). In some embodiments,a polynucleotide is isolated or purified.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other material,e.g., cellular material, culture medium, other nucleic acid molecules,chemical precursors and/or other chemicals.

In particular aspects, described herein are polynucleotides comprisingnucleotide sequences encoding antibodies or antigen-binding fragmentsthereof, which immunospecifically bind to a MERTK polypeptide (e.g.,human MERTK) and comprises an amino acid sequence as described herein,as well as antibodies which compete with such antibodies for binding toa MERTK polypeptide (e.g., in a dose-dependent manner), or which bindsto the same or an overlapping epitope as that of such antibodies.

In certain aspects, described herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a heavy chain comprising the VH CDRs described herein(see, e.g., Tables 1 and 3). The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL CDRs described herein(see, e.g., Tables 2 and 4).

In particular embodiments, described herein are polynucleotidescomprising a nucleotide sequence encoding a MERTK agonistic antibodycomprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, andVH CDR3 as described in Table 1 or Table 3. In specific embodiments,described herein are polynucleotides comprising a nucleotide sequenceencoding a MERTK agonistic antibody comprising three VL chain CDRs,e.g., containing VL CDR1, VL CDR2, and VL CDR3 as described in Table 2or Table 4. In specific embodiments, described herein arepolynucleotides comprising a nucleotide sequence encoding a MERTKagonistic antibody comprising three VH chain CDRs, e.g., containing VHCDR1, VH CDR2, and VH CDR3 as described in Table 1 or Table 3, and threeVL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 asdescribed in Table 2 or Table 4. In specific embodiments, apolynucleotide described herein encodes a VH CDR1, a VH CDR2, and a VHCDR3 of M6 (i.e., SEQ ID NOs: 1, 6, and 11; SEQ ID NOs: 2, 7, and 12;SEQ ID NOs: 3, 8, and 11; SEQ ID NOs: 4, 9, and 13; SEQ ID NOs: 5, 10,and 14; or SEQ ID NOs: 3, 6, and 11). In specific embodiments, apolynucleotide described herein encodes a VL CDR1, a VL CDR2, and a VLCDR3 of M6 (i.e., SEQ ID NOs: 15, 19, and 22; SEQ ID NOs: 16, 20, and23; SEQ ID NOs: 17, 21, and 24; or SEQ ID NOs: 18, 20, and 22). Inspecific embodiments, a polynucleotide described herein encodes the a VHCDR1, a VH CDR2, and a VH CDR3 of M6 (i.e., SEQ ID NOs: 1, 6, and 11;SEQ ID NOs: 2, 7, and 12; SEQ ID NOs: 3, 8, and 11; SEQ ID NOs: 4, 9,and 13; SEQ ID NOs: 5, 10, or 14; or SEQ ID NOs: 3, 6, and 11), and thethree VL CDRs of M6 (i.e., SEQ ID NOs: 15, 19, and 22; SEQ ID NOs: 16,20, and 23; SEQ ID NOs: 17, 21, and 24; or SEQ ID NOs: 18, 20, and 22).

In specific embodiments, a polynucleotide described herein encodes a VHCDR1, a VH CDR2, and a VH CDR3 of M19 (i.e., SEQ ID NOs: 25, 30, and 35;SEQ ID NOs: 26, 31, and 36; SEQ ID NOs: 27, 32, and 35; SEQ ID NOs: 28,33, and 37; SEQ ID NOs: 29, 34, and 38; or SEQ ID NOs: 27, 30, and 35).In specific embodiments, a polynucleotide described herein encodes a VLCDR1, a VL CDR2, and a VL CDR3 of M19 (e.g., SEQ ID NOs: 39, 43, and 46;SEQ ID NOs: 40, 44, and 47; SEQ ID NOs: 41, 45, and 48; or SEQ ID NOs:42, 44, and 46). In specific embodiments, a polynucleotide describedherein encodes the three VH CDRs of M19 (i.e., SEQ ID NOs: 25, 30, and35; SEQ ID NOs: 26, 31, and 36; SEQ ID NOs: 27, 32, and 35; SEQ ID NOs:28, 33, or 37; SEQ ID NOs: 29, 34, and 38; or SEQ ID NOs: 27, 30, and35), and the three VL CDRs of M19 (i.e., SEQ ID NOs: 39, 43, and 46; SEQID NOs: 40, 44, and 47; SEQ ID NOs: 41, 45, and 48; or SEQ ID NOs: 42,44, and 46).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an anti-MERTK antibody described hereincomprising a heavy chain variable region that comprises an amino acidsequence described herein (SEQ ID NO: 49 or 51), wherein the antibodyimmunospecifically binds to MERTK (e.g., human MERTK, or both human andmouse MERTK). In certain embodiments, a polynucleotide described hereincomprises a nucleotide sequence encoding an anti-MERTK antibodydescribed herein comprising a light chain variable region that comprisesan amino acid sequence described herein (SEQ ID NOs: 50 or 52), whereinthe antibody immunospecifically binds to MERTK (e.g., human MERTK, orboth human and mouse MERTK). In certain embodiments, a polynucleotidedescribed herein comprises a nucleotide sequence encoding an anti-MERTKantibody described herein comprising a heavy chain variable region thatcomprises the amino acid sequence of SEQ ID NO: 49, and a light chainvariable region that comprises the amino acid sequence of SEQ ID NO: 50,wherein the antibody immunospecifically binds to MERTK (e.g., humanMERTK, or both human and mouse MERTK). In certain embodiments, apolynucleotide described herein comprises a nucleotide sequence encodingan anti-MERTK antibody described herein comprising a heavy chainvariable region that comprises the amino acid sequence of SEQ ID NO: 51,and a light chain variable region that comprises the amino acid sequenceof SEQ ID NO: 52, wherein the antibody immunospecifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK).

In certain embodiments, a polynucleotide described herein encodes a VHcomprising a nucleic acid sequence of SEQ ID NO: 53 or SEQ ID NO: 55. Incertain embodiment, a polynucleotide described herein encodes a VLcomprising a nucleic acid sequence of SEQ ID NO: 54 or SEQ ID NO: 56. Ina specific embodiment, a polynucleotide described herein encodes a VHcomprising a nucleic acid sequence of SEQ ID NO: 53, and a VL comprisingthe nucleic acid sequence of SEQ ID NO: 54 (e.g., M6). In anotherspecific embodiment, a polynucleotide described herein encodes a VHcomprising a nucleic acid sequence of SEQ ID NO: 55, and a VL comprisingthe nucleic acid sequence of SEQ ID NO: 56 (e.g., M19).

In specific aspects, described herein is a polynucleotide comprising anucleotide sequence encoding an antibody comprising a light chain and aheavy chain, e.g., a separate light chain and heavy chain. With respectto the light chain, in a specific embodiment, a polynucleotide describedherein comprises a nucleotide sequence encoding a kappa light chain. Inanother specific embodiment, a polynucleotide described herein comprisesa nucleotide sequence encoding a lambda light chain. In yet anotherspecific embodiment, a polynucleotide described herein comprises anucleotide sequence encoding an antibody described herein comprising ahuman kappa light chain or a human lambda light chain. In a particularembodiment, a polynucleotide described herein comprises a nucleotidesequence encoding an antibody described herein, which immunospecificallybinds to MERTK (e.g., human MERTK, or both human and mouse MERTK) andagonizes MERTK signaling of endothelial cells wherein the antibodycomprises a light chain, and wherein the amino acid sequence of thevariable region of the light chain can comprise any amino acid sequenceof SEQ ID NO: 50 or SEQ ID NO: 52, and wherein the constant region ofthe light chain comprises the amino acid sequence of a human kappa lightchain constant region. In another particular embodiment, apolynucleotide described herein comprises a nucleotide sequence encodingan antibody described herein, which immunospecifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTKsignaling of endothelial cells and comprise a light chain, wherein theamino acid sequence of the variable region of the light chain cancomprise any amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52, andwherein the constant region of the light chain comprises the amino acidsequence of a human lambda light chain constant region.

In certain embodiments, an optimized polynucleotide sequence encoding anMERTK agonistic antibody described herein or an antigen-binding fragmentthereof (e.g., VH domain and/or VL domain) can hybridize to an antisense(e.g., complementary) polynucleotide of an unoptimized polynucleotidesequence encoding an MERTK agonistic antibody described herein or anantigen-binding fragment thereof (e.g., VH domain and/or VL domain). Inspecific embodiments, an optimized nucleotide sequence encoding an MERTKagonistic antibody described herein or an antigen-binding fragmenthybridizes under high stringency conditions to antisense polynucleotideof an unoptimized polynucleotide sequence encoding an MERTK agonisticantibody described herein or an antigen-binding fragment thereof. In aspecific embodiment, an optimized nucleotide sequence encoding a MERTKagonistic antibody described herein or an antigen-binding fragmentthereof hybridizes under high stringency, intermediate or lowerstringency hybridization conditions to an antisense polynucleotide of anunoptimized nucleotide sequence encoding an MERTK agonistic antibodydescribed herein or an antigen-binding fragment thereof. In a specificembodiment, a polynucleotide described herein is codon optimized.Information regarding hybridization conditions has been described, see,e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g.,paragraphs 72-73), which is incorporated herein by reference.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, and modified versions ofthese antibodies can be determined using methods well known in the art,i.e., nucleotide codons known to encode particular amino acids areassembled in such a way to generate a nucleic acid that encodes theantibody. Such a polynucleotide encoding the antibody can be assembledfrom chemically synthesized oligonucleotides (e.g., as described inKutmeier G et al., (1994), BioTechniques 17: 242-6), which, briefly,involves the synthesis of overlapping oligonucleotides containingportions of the sequence encoding the antibody, annealing and ligatingof those oligonucleotides, and then amplification of the ligatedoligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding MERTK agonistic antibodies described herein can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the MERTK agonistic antibodies).Hybridoma cells can serve as a source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of MERTK agonistic antibodies in the recombinanthost cells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise a promoter, a secretion signal, a cloning site for thevariable domain, constant domains, and a selection marker. The VH and VLdomains can also be cloned into one vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also described are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH (SEQ ID NO: 49 or 51) and/or VL (SEQ IDNO: 50 or 52) provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

In specific embodiments, the drug moiety is a peptide or protein fusedto the N-terminus or C-terminus of one or more chains of the antibodymoiety (directly or via a linker that is a peptide or protein). Forexample, when the antibody moiety is an scFv, the drug moiety can befused at the N- or C-terminus of the scFv (directly or via a linker thatis a peptide or protein). In a specific embodiment, when the antibodymoiety is a multi-chain antibody or antigen-binding fragment thereof,the drug moiety can be fused to one of the chains of the antibody moiety(directly or via a linker that is a peptide or protein). In the case ofan antibody that is an immunoglobulin, in a specific embodiment, bothheavy chains can be fused to the drug moiety (directly or via a linkerthat is a peptide or protein), and/or both light chains can be fused tothe linker drug moiety (directly or via a linker that is a peptide orprotein). In such specific embodiments, provided herein arepolynucleotides comprising a nucleotide sequence encoding a fusionprotein composed of the drug moiety and the antibody moiety or a chainthereof, and the linker (if there is one) between the drug moiety andthe antibody moiety. Such polynucleotides can be generated and isolatedusing the same methods as described above for anti-MERTK antibodies.

5.2.3. Cells and Vectors

Also described herein are vectors (e.g., expression vectors) comprisingpolynucleotides comprising nucleotide sequences encoding MERTK agonisticantibodies or an antigen-binding fragment thereof for recombinantexpression in host cells, preferably in mammalian cells. Also describedherein are host cells comprising such vectors for recombinantlyexpressing MERTK agonistic antibodies or antigen-binding fragmentsthereof described herein (e.g., human or humanized antibody).

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to MERTK(e.g., human MERTK, or both human and mouse MERTK) involves constructionof an expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule, heavyand/or light chain of an antibody, or an antigen-binding fragmentthereof (e.g., heavy and/or light chain variable regions) describedherein has been obtained, the vector for the production of the antibodymolecule can be produced by recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing a protein byexpressing a polynucleotide containing an antibody or antibody fragment(e.g., light chain or heavy chain) encoding nucleotide sequence aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibody orantibody fragment (e.g., light chain or heavy chain) coding sequencesand appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Also describedare replicable vectors comprising a nucleotide sequence encoding anantibody molecule described herein, a heavy or light chain of anantibody, a heavy or light chain variable domain of an anti-MERTKantibody or antigen-binding fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464) and variable domains ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or anantigen-binding fragment thereof. Thus, described herein are host cellscontaining a polynucleotide encoding an antibody described herein or anantigen-binding fragment thereof, or a heavy or light chain thereof, ora fragment thereof, or a single chain antibody described herein,operably linked to a promoter for expression of such sequences in thehost cell. As used herein, the term “host cell” can be any type of cell,e.g., a primary cell, a cell in culture, or a cell from a cell line. Inspecific embodiments, the term “host cell” refers to a cell transfectedwith a nucleic acid molecule and the progeny or potential progeny ofsuch a cell. Progeny of such a cell may not be identical to the parentcell transfected with the nucleic acid molecule, e.g., due to mutationsor environmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein. Such host-expression systemsrepresent vehicles by which the coding sequences of interest can beproduced and subsequently purified, but also represent cells which can,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule described herein in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems (e.g., green algae such as Chlamydomonasreinhardtii) infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6,VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210,R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific embodiment, cells for expressingantibodies described herein or an antigen-binding fragment thereof areCHO cells, for example CHO cells from the CHO GS System™ (Lonza). In aparticular embodiment, cells for expressing antibodies described hereinare human cells, e.g., human cell lines. In a specific embodiment, amammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particularembodiment, bacterial cells such as Escherichia coli, or eukaryoticcells (e.g., mammalian cells), especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-5; and Cockett M I etal., (1990) Biotechnology 8(7): 662-7). In certain embodiments,antibodies described herein are produced by CHO cells or NS0 cells. In aspecific embodiment, the expression of nucleotide sequences encodingantibodies described herein which immunospecifically bind MERTK (e.g.,human MERTK, or both human and mouse MERTK) and agonizes MERTK signalingof endothelial cells is regulated by a constitutive promoter, induciblepromoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like. For example, pGEXvectors can also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals canalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544).

In addition, a host cell strain which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired can be chosen. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, MERTK agonistic antibodiesdescribed herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein orantigen-binding fragments thereof have reduced fucose content or nofucose content. Such antibodies can be produced using techniques knownone skilled in the art. For example, the antibodies can be expressed incells deficient or lacking the ability of to fucosylate. In a specificexample, cell lines with a knockout of both alleles ofa1,6-fucosyltransferase can be used to produce antibodies orantigen-binding fragments thereof with reduced fucose content. ThePotelligent® system (Lonza) is an example of such a system that can beused to produce antibodies or antigen-binding fragments thereof withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an MERTK agonistic antibody described herein or anantigen-binding fragment thereof can be engineered. In specificembodiments, a cell described herein stably expresses a lightchain/light chain variable domain and a heavy chain/heavy chain variabledomain which associate to form an antibody described herein or anantigen-binding fragment thereof.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an MERTK agonistic antibodydescribed herein or an antigen-binding fragment thereof. Such engineeredcell lines can be particularly useful in screening and evaluation ofcompositions that interact directly or indirectly with the antibodymolecule.

A number of selection systems can be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56). Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse G F et al., (1983) Mol Cell Biol3: 257-66).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In such anexpression vector, the transcription of both genes can be driven by acommon promoter, whereas the translation of the mRNA from the first genecan be by a cap-dependent scanning mechanism and the translation of themRNA from the second gene can be by a cap-independent mechanism, e.g.,by an IRES.

In specific embodiments, the drug moiety is a peptide or protein fusedto the N-terminus or C-terminus of one or more chains of the antibodymoiety (directly or via a linker that is a peptide or protein). Forexample, when the antibody moiety is an scFv, the drug moiety can befused at the N- or C-terminus of the scFv (directly or via a linker thatis a peptide or protein). In a specific embodiment, when the antibodymoiety is a multi-chain antibody or antigen-binding fragment thereof,the drug moiety can be fused to one of the chains of the antibody moiety(directly or via a linker that is a peptide or protein). In the case ofan antibody that is an immunoglobulin, in a specific embodiment, bothheavy chains can be fused to the drug moiety (directly or via a linkerthat is a peptide or protein), and/or both light chains can be fused tothe drug moiety (directly or via a linker that is a peptide or protein).In such specific embodiments, provided herein are vectors (e.g.,expression vectors) comprising polynucleotides comprising a nucleotidesequence encoding a fusion protein composed of the drug moiety and theantibody moiety or a chain thereof, and the linker (if there is one)between the drug moiety and the antibody moiety, for recombinantexpression in host cells, preferably in mammalian cells. Also providedherein are ex vivo host cells comprising such vectors for recombinantlyexpressing the fusion protein composed of the drug moiety and theantibody moiety or a chain thereof, and the linker (if there is one)between the drug moiety and the antibody moiety. Characteristcis of andmethods for generating such vectors and ex vivo host cells can be thesame as described above for anti-MERTK antibodies. Also provided hereinare methods of producing the fusion protein composed of the drug moietyand the antibody moiety or a chain thereof, and the linker (if there isone) between the drug moiety and the antibody moiety, comprisingculturing such an ex vivo host cell under conditions such that thepolynucleotide comprising a nucleotide sequence encoding the fusionprotein is expressed by the ex vivo host cell to produce the fusionprotein.

5.3. Functional Characteristics of the Antibody-Drug Conjugates

In certain embodiments, an antibody-drug conjugate provided hereincauses cell death of MERTK-expressing endothelial cells in vitro. Insome specific embodiments, at least 10%, 20%, 30%, 35%, 40%, 50%, or 60%of MERTK-expressing endothelial cells cultured for a period of time inthe presence of an antibody-drug conjugate described herein are dead atthe end of the period of time, as assessed by methods known to one ofskill in the art for measuring cell death in vitro. In other specificembodiments, the percentage of MERTK-expressing endothelial cells thatundergo cell death is at least 50%, 100%, 2-fold, 3-fold, 5-fold,10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-foldhigher when cultured for a period of time in the presence of anantibody-drug conjugate described herein, relative to when culturedwithout the antibody-drug conjugate (e.g., cultured without anyantibody-drug conjugate or cultured with an antibody-drug conjugatecomprising an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to MERTK) or cultured with an unconjugatedanti-MERTK antibody or unconjugated anti-MERTK antigen-binding fragmentdescribed herein). The period of time can be, for example, about 1 hr, 2hrs, 4 hrs, 8 hrs, 12 hrs, 16 hrs, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or a week.

In specific embodiments, an antibody-drug conjugate described hereininhibits angiogenesis within tumors. In some embodiments, the inhibitionof angiogenesis is by at least 10%, 20%, 30%, 40%, 50%, 55, 60%, 65%,70%, or 75%. In a specific embodiment, the inhibition of angiogenesis isat least 50%, 55%, 60%, 65%, or 70%. Inhibition of angiogenesis can beassessed by methods described herein and/or known to one of skill in theart. The inhibition can be relative to the level of angiogenesis withoutthe antibody-drug conjugate (e.g., cultured without any antibody-drugconjugate or cultured with an antibody-drug conjugate comprising anunrelated antibody (e.g., an antibody that does not immunospecificallybind to MERTK) or cultured with an unconjugated anti-MERTK antibody orunconjugated anti-MERTK antigen-binding fragment described herein). In aspecific embodiment, the assay that is used to assess angiogenesis invivo is as described in Example 6, infra.

In certain embodiments, an antibody-drug conjugate described hereininhibits tumor (e.g., human breast cancer tumor) progression. Theinhibition of tumor progression by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, or 80%. Tumor progression can be assessed by methods describedherein and/or known to one of skill in the art. The tumor progressioncan be relative to the cancer status without the antibody-drug conjugate(e.g., cultured without any antibody-drug conjugate or cultured with anantibody-drug conjugate comprising an unrelated antibody (e.g., anantibody that does not immunospecifically bind to MERTK) or culturedwith an unconjugated anti-MERTK antibody or unconjugated anti-MERTKantigen-binding fragment described herein). In a specific embodiment,the assay that is used to assess tumor progression is a murine tumortransplantation model as described in Examples 5 and 8, infra.

5.4. The Drug Moiety and the Linker

The cytotoxic agent used in antibody-drug conjugates is also oftencalled a payload or warhead. Most of the cytotoxic agents used inantibody-drug conjugates target DNA or microtubules and have highpotency of cytotoxicity (with an IC₅₀ range of approximately 10⁻¹⁰-10⁻¹²M) (see Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337). Thecytotoxic agent can be a small molecule, a nucleotide, a peptide, or anon-antibody protein. In a specific embodiment, the cytotoxic agent is asmall molecule. In another specific embodiment, the cytotoxic agent is anon-antibody protein. Non-limiting exemplary cytotoxic agents that canbe used in the antibody-drug conjugates according to the invention aredescribed in Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337;Peters C and Brown S, (2015) Biosci Rep 35: art:e00225; McCombs J R andOwen S C (2015) The AAPS Journal 17: 339-351; Jackson D Y (2016) OrgProcess Res Dev 20: 852-866; and Olivier K J and Hurvitz S A ed., (2016)Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical,Wiley. In specific embodiments, the cytotoxic agent used in theantibody-drug conjugate is an auristatin (such as monomethyl auristatinE (MMAE), monomethyl auristatin F (MMAF), Aur0101, PF06380101,Auristatin W, or auristatin F) or derivative thereof, a maytansinoid(such as DM1 or DM4), a pyrrolobenzodiazepine (PBD) (such as SGD1882 orSG3199), an indolinobenzodiazepine (such as DGN462 or DGN549), acalicheamicin (ozogamicin) (such as CM1), a camptothecin analogue (suchas SN38, DX-8951f, or DX-8951f derivative), a duocarmycin (such asseco-duocarmycin-hydroxy-benzamide-azaindole (seco-DUBA), minorgroove-binding alkylating agent (MGBA), or MED-2460), a tubulininhibitor (such as cryptophycin), a tubulysin or tubulysin analogue(such as AZ13599185), amberstatin269, doxorubicin, an antibiotic (suchas rifalogue), an anthracycline (such as PNU-159682), a microtubuleinhibitor (such as rhizoxin), a spliceostatin, or a thailanstatin. In aspecific embodiment, the cytotoxic agent used in the antibody-drugconjugate is MMAE or MA/IAF. In another specific embodiment, thecytotoxic agent used in the antibody-drug conjugate is DM1 or DM4.

As used herein, the term “small molecule” refers to an organic orinorganic compound (which can be, for example, a heteroorganic ororganometallic compound) having a molecular weight of less than about10,000 grams per mole. In specific embodiments, the small molecule has amolecular weight of less than about 5,000 grams per mole, or less thanabout 2,000 grams per mole, or less than about 1,000 grams per mole, orless than about 500 grams per mole, or less than about 100 grams permole.

In some embodiments, the cytotoxic agent is conjugated directly to theantibody moiety. In other embodiments, the cytotoxic agent is conjugatedto the antibody moiety via a linker. Appropriate linkers to be usedaccording to the invention preferably are stable in the blood stream tolimit off-target toxicity and labile at the cancer site to allow forrelease of the cytotoxic agent (see Peters C and Brown S, (2015) BiosciRep 35: art:e00225). Non-limiting exemplary linkers that can be usedaccording to the invention are described in Beck A et al., (2017) NatRev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; and OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. In some embodiments, the linkeris a cleavable linker, for example, having a motif sensitive to alysosomal protease (for example, cathepsin B), a motif sensitive to anacidic pH (for example, hydrazone), or a motif containing a disulfidebridge that can be reduced by glutathione. In other embodiments, thelinker is a non-cleavable linker. By way of example but not limitation,the linker can be a small molecule, a nucleotide, a peptide, or anon-antibody protein. In a specific embodiment, the linker is a smallmolecule. In a specific embodiment, the linker used in the antibody-drugconjugate is cathepsin B, hydrazone,succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),maleimidocaproic acid (mc), valine-citrulline (vc),N-hydroxysuccinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB),N-hydroxysuccinimidyl 4-(2-pyridydithio)butanoate (SPDB), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethyleneglycol 8-valine-citrulline (PEG8-va), mb-vc, CL2A, cleavable vc-basedlinker, or fleximer polymer linker.

Non-limiting exemplary linker-drug moiety pairs that can be usedaccording to the invention are described in Beck A et al., (2017) NatRev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; and OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. In specific embodiments, thelinker-drug moiety pair in the antibody-drug conjugate is vc-MMAE,mc-MMAF, SMCC-DM1, sulfo-SPDB-DM4, SPDB-DM4, SPP-DM1, va-SGD1882,polyethylene glycol 8 (PEG8)-va-SG3199, sulfo-SPDB-DGN462,hydrazone-CM1, vc-seco-DUBA, mb-vc-MGBA, CL2A-SN38, peptide linker withDX-8951 derivative, hydrazone-doxorubicin, cleavable vc-based linkerwith Aur0101, vc-PF06380101, fleximer polymer linker with auristatin F,cleavable linker-tubulin inhibitor, or vc-rifalogue.

5.5. Production of the Antibody-Drug Conjugates

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein the linker is notpresent, the method comprising: (a) conjugating the cytotoxic agentdirectly to the antibody moiety to produce the antibody-drug conjugate;and (b) purifying the antibody-drug conjugate.

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein the antibody-drugconjugate comprises the linker, the method comprising the followingsteps in the order stated: (a) conjugating the linker directly to theantibody moiety to produce a linker-antibody moiety; (b) conjugating thelinker of the linker-antibody moiety directly to the cytotoxic agent toproduce the antibody-drug conjugate; and (c) purifying the antibody-drugconjugate.

In another aspect, provided herein are methods of producing anantibody-drug conjugate described herein wherein the antibody-drugconjugate comprises the linker, the method comprising the followingsteps in the order stated: (a) conjugating the linker directly to thecytotoxic agent to produce a linker-cytotoxic agent moiety; (b)conjugating the linker of the linker-cytotoxic agent moiety directly tothe antibody moiety to produce the antibody-drug conjugate; and (c)purifying the antibody-drug conjugate.

The conjugating and purifying steps can be performed by methods known inthe art used for producing antibody-drug conjugates, such as the methodsdescribed in Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337;Peters C and Brown S, (2015) Biosci Rep 35: art:e00225; McCombs J R andOwen S C (2015) The AAPS Journal 17: 339-351; Jackson D Y (2016) OrgProcess Res Dev 20: 852-866; or Olivier K J and Hurvitz S A ed., (2016)Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical,Wiley. In some embodiments, the drug moiety is conjugated to one chainof the antibody moiety (for example, when the antibody moiety is a scFv,or when the antibody moiety is a multi-chain antibody, such as animmunoglobulin (which is a tetramer), or antigen-binding fragmentthereof). In other embodiments, the drug moiety is conjugated to two ormore chains of the antibody moiety (when the antibody moiety is amulti-chain antibody, such as an immunoglobulin, or antigen-bindingfragment thereof). In a specific embodiment, the drug moiety isconjugated to two identical chains of an immunoglobulin, e.g., the heavychains or the light chains. In other embodiments, the drug moiety isconjugated to all chains of the antibody moiety (when the antibodymoiety is a multi-chain antibody, such as an immunoglobulin orantigen-binding fragment thereof). In a specific embodiment, theantibody-drug conjugate described herein is purified by chromatograph.In another specific embodiment, the antibody-drug conjugate describedherein is purified by centrifugation. By way of example but notlimitation, the conjugation chemistry that can be used for generatingthe antibody-drug conjugate can be thiol plus maleimide, thiol plusself-hydrolyzing maleimide, thiol plus phenyloxadiazole sulfone, oximeligation, alkoxyamine-to-keto-group reaction, strain-promotedazide-alkyne cycloaddition (SPAAC), copper-free click chemistry,cysteine oxidized to formylglycine, hydrazino-iso-Pictet-Spengler (HIPS)ligation, ligation of γ-carboxyamide group from glutamine residues plusprimary amines, ligation LPETG plus primary amine of polyglycine motif,maleimide plus 6-thiofucose, fucose-specific conjugation of hydrazide,periodate oxidation (aldehyde) plus amino-oxy-payload, strain-promotedalkyn-azide cycloaddition, C2-keto-gal oximation, site selectivealdehyde oxidation plus oxime ligation, oxime ligation NH2 plusindole-based 5-difluoro-2,4-dinitrobenzene derivatives, thiol plusbis-sulfone, thiol plus dibromomaleimide, thiol plus maleimide followedby pH 9.2 treatment (45° C., 48 hours), or thiol plus arylpropionitrile(see Beck A et al., (2017) Nat Rev Drug Discov 16: 315-337).

The anti-MERTK antibody or an antigen-binding fragment thereof describedherein can be generated as described in Section 5.2. In specificembodiments, the anti-MERTK antibody or an antigen-binding fragmentthereof described herein is engineered or modified by a method known inthe art to facilitate conjugation with the drug moiety(ies), inparticular to facilitate site-specific conjugation with the drugmoiety(ies), for example, by a method described in Beck A et al., (2017)Nat Rev Drug Discov 16: 315-337; Peters C and Brown S, (2015) Biosci Rep35: art:e00225; McCombs J R and Owen S C (2015) The AAPS Journal 17:339-351; Jackson D Y (2016) Org Process Res Dev 20: 852-866; or OlivierK J and Hurvitz S A ed., (2016) Antibody-Drug Conjugates: Fundamentals,Drug Development, and Clinical, Wiley. Non-limiting exemplary methodsthat can be used (see Beck A et al., (2017) Nat Rev Drug Discov 16:315-337) to engineer or modify an anti-MERTK antibody or anantigen-binding fragment thereof to facilitate conjugation with the drugmoiety(ies) include adding one or more additional cysteines orselenocysteines, unnatural amino acid engineering, adding one or moreamino acid tags recognizable by certain enzymes that can assist with theconjugation, glycan remodeling, adding an amino-terminal serine, andnative cysteine bridging.

The linker and the cytotoxic agent can be generated by any method knownin the art for producing the linker and the cytotoxic agent,respectively.

5.6. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising anantibody-drug conjugate described herein and a pharmaceuticallyacceptable carrier. In a specific embodiment, the antibody-drugconjugate is purified. In a specific embodiment, the antibody-drugconjugate is present in the pharmaceutical composition in atherapeutically effective amount.

In a specific embodiment, the population of antibody-drug conjugatescontained in the pharmaceutical composition have a drug-to-antibodyratio (DAR) between 1 to 20. DAR is the average number of cytotoxicagents conjugated to the antibodies. In another specific embodiment, thepopulation of antibody-drug conjugates contained in the pharmaceuticalcomposition have a DAR between 1 to 15. In another specific embodiment,the population of antibody-drug conjugates contained in thepharmaceutical composition have a DAR between 1 to 12. In anotherspecific embodiment, the population of antibody-drug conjugatescontained in the pharmaceutical composition have a DAR between 1 to 8.In another specific embodiment, the population of antibody-drugconjugates contained in the pharmaceutical composition have a DARbetween 3 to 5. In another specific embodiment, the population ofantibody-drug conjugates contained in the pharmaceutical compositionhave a DAR between 3.5 to 4.

Acceptable carriers, which can be excipients or stabilizers, arenontoxic to recipients at the dosages and concentrations employed, andinclude but are not limited to buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise anantibody-drug conjugate described herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In a specific embodiment, pharmaceuticalcompositions comprise an effective amount of an antibody-drug conjugatedescribed herein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In aspecific embodiment, the pharmaceutical compositions and/orantibody-drug conjugate described herein, can be combined with atherapeutically effective amount of any of the additional therapeuticagents described herein (See Section 5.7.2, infra).

In some embodiments, the antibody-drug conjugate is the only activeingredient included in the pharmaceutical composition.

Pharmaceutical compositions described herein can be used to treatcancer.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody-drug conjugateinclude sterile solutions ready for injection, sterile dry solubleproducts, such as lyophilized powders, ready to be combined with asolvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions. Thesolutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody-drug conjugate are prepared asdescribed for the local and systemic administration. The resultingmixture can be a solution, suspension, emulsions or the like and can beformulated as creams, gels, ointments, emulsions, solutions, elixirs,lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations,sprays, suppositories, bandages, dermal patches or any otherformulations suitable for topical administration.

An antibody-drug conjugate described herein can be formulated as anaerosol for topical application, such as by inhalation (see, e.g., U.S.Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosolsfor delivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microtine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

An antibody-drug conjugate described herein can be formulated for localor topical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracisternal orintraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the antibody-drugconjugate alone or in combination with other pharmaceutically acceptableexcipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody-drug conjugate. For example, such patches aredisclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301,6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

In certain embodiments, a pharmaceutical composition comprising anantibody-drug conjugate described herein is a lyophilized powder, whichcan be reconstituted for administration as solutions, emulsions andother mixtures. It may also be reconstituted and formulated as solids orgels. The lyophilized powder is prepared by dissolving an antibody-drugconjugate described herein, or a pharmaceutically acceptable derivativethereof, in a suitable solvent. In some embodiments, the lyophilizedpowder is sterile. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The antibody-drug conjugates described herein and other compositionsprovided herein can also be formulated to be targeted to a particulartissue, receptor, or other area of the body of the subject to betreated. Many such targeting methods are well known to those of skill inthe art. All such targeting methods are contemplated herein for use inthe instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874. In a specific embodiment, anantibody-drug conjugate described herein is targeted to a tumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.7. Uses and Methods

5.7.1. Therapeutic Uses and Methods

5.7.1.1. Cancer

In one aspect, presented herein are methods for treating cancer in asubject, comprising administering to a subject in need thereof anantibody-drug conjugate described herein, or a pharmaceuticalcomposition comprising an antibody-drug conjugate described herein. In aspecific embodiment, presented herein are methods for treating cancer ina subject, comprising administering to a subject in need thereof apharmaceutical composition comprising an antibody-drug conjugatedescribed herein. In a particular embodiment, presented herein aremethods for treating cancer in which it is desirable to agonize MERTKsignaling of endothelial cells, for example to inhibit angiogenesis,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising an antibody-drug conjugate described herein.

In specific embodiments, the administration of an antibody-drugconjugate described herein, or a pharmaceutical composition describedherein to a subject with cancer achieves at least one, two, three, fouror more of the following effects: (i) the reduction or amelioration ofthe severity of one or more symptoms of cancer; (ii) the reduction inthe duration of one or more symptoms associated with cancer; (iii) theprevention in the recurrence of a symptom associated with cancer; (iv)the reduction in hospitalization of a subject; (v) a reduction inhospitalization length; (vi) the increase in the survival of a subject;(vii) the enhancement or improvement of the therapeutic effect ofanother therapy; (viii) the inhibition of the development or onset ofone or more symptoms associated with cancer; (ix) the reduction in thenumber of symptoms associated with cancer; (x) improvement in quality oflife as assessed by methods well known in the art; (x) inhibition of therecurrence of a tumor; (xi) the regression of tumors and/or one or moresymptoms associated therewith; (xii) the inhibition of the progressionof tumors and/or one or more symptoms associated therewith; (xiii) areduction in the growth of a tumor; (xiv) a decrease in tumor size(e.g., volume or diameter); (xv) a reduction in the formation of a newlyformed tumor; (xvi) prevention, eradication, removal, or control ofprimary, regional and/or metastatic tumors; (xvii) a decrease in thenumber or size of metastases; (xviii) a reduction in mortality; (xix) anincrease in relapse free survival; (xx) the size of the tumor ismaintained and does not increase or increases by less than the increaseof a tumor after administration of a standard therapy as measured byconventional methods available to one of skill in the art, such asmagnetic resonance imaging (MRI), dynamic contrast-enhanced MRI(DCE-MRI), X-ray, and computed tomography (CT) scan, or a positronemission tomography (PET) scan; and/or (xxi) an increase in the lengthof remission in patients.

In some embodiments, the cancer treated in accordance with the methodsdescribed herein is a cancer of the lung, breast, bone, ovary, stomach,pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate orthyroid. In some embodiments, the cancer is a sarcoma, squamous cellcarcinoma, melanoma, glioma, glioblastoma, neuroblastoma or Kaposi'ssarcomas. In some embodiments, the cancer treated in accordance with themethods is metastatic.

In a specific embodiment, the cancer treated in accordance with themethods described herein is breast cancer. In a particular embodiment,the cancer treated in accordance with the methods described herein istriple-negative breast cancer. Triple-negative breast cancer refers toany tumor or cell derived from or growing in breast tissue that does notexpress the genes Her2 (also known as Neu), Estrogen Receptor (alsoknown as ER) or Progesterone Receptor (also known as PR).

As used herein, the terms “subject” and “patient” are usedinterchangeably. In some embodiments, the subject is a mammal such as aprimate (e.g., monkey or human), most preferably a human.

In a specific embodiment, an antibody-drug conjugate described herein isadministered to a subject. In certain embodiments, two or more differentantibody-drug conjugates described herein are administered to a subject.In some embodiments, an antibody-drug conjugate described herein isadministered to a subject in combination with one or more othertherapies (See Section 5.7.2, Infra.).

5.7.1.2. Routes of Administration and Dosage

An antibody-drug conjugate, or composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intratumoral, conjunctival and subcutaneous routes.Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent for useas a spray. In one embodiment, an antibody-drug conjugate, or acomposition described herein is administered parenterally to a subject.In a specific embodiment, said parenteral administration is intravenous,intramuscular, or subcutaneous. In a specific embodiment, anantibody-drug conjugate is administered intravenously to a subject.

The amount of an antibody-drug conjugate, or composition which will beeffective in the treatment and/or prevention of a condition will dependon the nature of the disease, and can be determined by standard clinicaltechniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the type of cancer, and should be decidedaccording to the judgment of the practitioner and each subject'scircumstances. For example, effective doses may also vary depending uponmeans of administration, target site, physiological state of the patient(including age, body weight and health), whether the patient is human oranimal, other medications administered, or whether treatment isprophylactic or therapeutic. Treatment dosages are optimally titrated tooptimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

For an antibody-drug conjugate, the dosage can range from about 0.0001to 100 mg/kg, and more usually 0.01 to 15 mg/kg, of the patient bodyweight. For example, dosages can be 1 mg/kg body weight, 10 mg/kg bodyweight, or within the range of 1-10 mg/kg or in other words, 70 mg or700 mg or within the range of 70-700 mg, respectively, for a 70 kgpatient. In some embodiments, the dosage administered to the patient isabout 1 mg/kg to about 20 mg/kg of the patient's body weight. Generally,human antibodies have a longer half-life within the human body thanantibodies from other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of antibody-drug conjugates comprisinghuman antibodies and less frequent administration is often possible.

An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. In somemethods, two or more antibody-drug conjugates are administeredsimultaneously to a subject. An antibody-drug conjugate is usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly, every 3 months, every 6 months or yearly.

5.7.2. Combination Therapies

In a specific embodiment, the methods provided herein for treatingcancer in a subject, comprising administering to a subject in needthereof a pharmaceutical composition comprising an antibody-drugconjugate described herein, further comprise administering to thesubject one or more additional therapeutic agents. In a specificembodiment, the additional therapeutic agent is for treating the cancer.In a specific embodiment, the additional therapeutic agent is fortreating any side effects of treatment with an antibody-drug conjugatedescribed herein.

In specific embodiments, the additional agent is an agent used to treatbreast cancer, an agent used to treat melanoma, an immunotherapy, or anangiogenesis inhibitor.

In a specific embodiment, the additional therapeutic agent is an agentused to treat breast cancer that is selected from the group consistingof Tamoxifen, Raloxifene, Paclitaxel (TAXOL®, Cyclophosphamide,Docetaxel, Vinblastine, Fluorouracil, Everolimus, Trastuzumab,Trastuzumab-Emtansine, Pertuzumab, and Lapatinib Ditosylate.

In a specific embodiment, the additional therapeutic agent is an agentused to treat melanoma that is selected from the group consisting of aBRAF inhibitor, a MEK inhibitor, and Dacarbazine.

In a specific embodiment, the additional therapeutic agent is anantibody that is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1inhibitor.

In a specific embodiment, the additional therapeutic agent is anangiogenesis inhibitor that is selected from the group consisting of aVEGF inhibitor, a VEGFR2 inhibitor, Sunitinib, and Sorafenib.

In other embodiments, the additional therapeutic agent is an agentlisted in Table 10.

TABLE 10 Additional Therapeutic Agents for Use in Combination Therapywith MERTK Antibodies or Antigen-Binding Fragments Thereof Alkylatingagents Busulfan Chlorambucil dacarbazine procarbazine ifosfamidealtretamine hexamethylmelamine estramustine phosphate thiotepamechlorethamine dacarbazine streptozocin lomustine temozolomidecyclophosphamide Semustine Platinum agents spiroplatin lobaplatin(Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464(Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo) ZD-0473 (AnorMED)AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolitesazacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorode-pentostatin oxyadenosine 6-mercaptopurine hydroxyurea 6-thioguaninedecitabine (SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxyirofulven (MGI Pharma) cytidine methotrexate DMDC (Hoffmann-La Roche)tomudex ethynylcytidine (Taiho) fludarabine gemcitabine RaltitrexedCapecitabine Topoisomerase amsacrine exatecan mesylate inhibitors(Daiichi) epirubicin quinamed (ChemGenex) etoposide gimatecan(Sigma-Tau) teniposide or diflomotecan mitoxantrone (Beaufour-Ipsen)7-ethyl-10-hydroxy- TAS-103 (Taiho) camptothecin dexrazoxanetelsamitrucin (Spectrum) (TopoTarget) pixantrone J-107088 (Merck & Co)(Novuspharma) rebeccamycin BNP-1350 (BioNumerik) analogue (Exelixis)BBR-3576 CKD-602 (Novuspharma) (Chong Kun Dang) rubitecan (SuperGen)KW-2170 (Kyowa Hakko) irinotecan (CPT-11) hydroxycamptothecin (SN-38)Topotecan Antitumor valrubicin azonafide antibiotics therarubicinanthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycinMEN-10755 (Menarini) porfiromycin GPX-100 (Gem Pharmaceuticals)mitoxantrone Epirubicin (novantrone) amonafide mitoxantrone DoxorubicinAntimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (CellTherapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCl) A 105972(Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin LU 223651(BASF) (Warner-Lambert) cemadotin (BASF) D 24851 (ASTAMedica) RPR109881A ER-86526 (Eisai) (Aventis) TXD 258 (Aventis) combretastatin A4(BMS) epothilone B isohomohalichondrin-B (Novartis) (PharmaMar) T 900607(Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi)cryptophycin 52 IDN-5109 (Indena) (Eli Lilly) vinflunine (Fabre) AVLB(Prescient NeuroPharma) auristatin PE azaepothilone B (BMS) (TeikokuHormone) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin(Protarga) CA-4 (OXiGENE) SB 408075 docetaxel (GlaxoSmithKline)Vinorelbine vincristine Trichostatin A Paclitaxel Aromataseaminoglutethimide YM-511 (Yamanouchi) inhibitors atamestane formestane(BioMedicines) letrozole exemestane anastrazole Thymidylate pemetrexed(Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331 (BTG)CoFactor ™ (BioKeys) DNA antagonists trabectedin edotreotide (Novartis)(PharmaMar) glufosfamide mafosfamide (Baxter International) (BaxterInternational) albumin + 32P apaziquone (Isotope Solutions) (SpectrumPharmaceuticals) thymectacin O6 benzyl guanine (NewBiotics) (Paligent)Farnesyltransferase arglabin tipifarnib inhibitors (NuOncology Labs)(Johnson & Johnson) lonafarnib perillyl alcohol (Schering-Plough) (DORBioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma)zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodardicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer)pivaloyloxymethyl acetyltransferase butyrate (Titan) inhibitors SAHA(Aton Pharma) depsipeptide (Fujisawa) MS-275 (Schering AG)Metalloproteinase Neovastat CMT-3 (CollaGenex) inhibitors (AeternaLaboratories) marimastat BMS-275291 (Celltech) (British Biotech)Ribonucleoside gallium maltolate tezacitabine (Aventis) reductaseinhibitors (Titan) triapine (Vion) didox (Molecules for Health) TNFalpha virulizin revimid (Celgene) agonists/antagonists (LorusTherapeutics) CDC-394 (Celgene) Endothelin A atrasentan (Abbott) YM-598(Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoic acidfenretinide alitretinoin (Ligand) receptor agonists (Johnson & Johnson)LGD-1550 (Ligand) Immuno- interferon dexosome therapy modulators(Anosys) oncophage pentrix (Australian Cancer (Antigenics) Technology)GMK (Progenics) ISF-154 (Tragen) adenocarcinoma cancer vaccine(Intercell) vaccine (Biomira) CTP-37 norelin (Biostar) (AVI BioPharma)IRX-2 (lmmuno-Rx) BLP-25 (Biomira) PEP-005 (Peplin MGV (Progenics)Biotech) synchrovax vaccines β-alethine (Dovetail) (CTL Immuno) melanomavaccine CLL therapy (Vasogen) (CTL Immuno) p21 RAS vaccine Ipilimumab(BMS), (GemVax) MAGE-A3 (GSK) CM-10 (cCam Biotherapeutics) nivolumab(BMS) MPDL3280A (Genentech) abatacept (BMS) pidilizumab (CureTech)lambrolizumab AMP-224 (GSK) (Merck) MEDI-4736 (AstraZeneca) Hormonal andestrogens dexamethasone antihormonal conjugated estrogens prednisoneagents ethinyl estradiol methylprednisolone chlortrianisen prednisoloneidenestrol aminoglutethimide hydroxyprogesterone leuprolide caproatemedroxyprogesterone octreotide testosterone mitotane testosterone P-04(Novogen) propionate; fluoxymesterone methyltestosterone2-methoxyestradiol (EntreMed) diethylstilbestrol arzoxifene (Eli Lilly)megestrol tamoxifen bicalutamide toremofine flutamide goserelinnilutamide Leuporelin bicalutamide Photodynamic talaporfinPd-bacteriopheophorbide agents (Light Sciences) (Yeda) Theralux lutetiumtexaphyrin (Theratechnologies) (Pharmacyclics) motexafin gadoliniumhypericin (Pharmacyclics) Kinase imatinib (Novartis) EKB-569 (Wyeth)Inhibitors leflunomide kahalide F (PharmaMar) (Sugen/Pharmacia) ZD1839CEP-701 (Cephalon) (AstraZeneca) erlotinib (Oncogene CEP-751 (Cephalon)Science) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera)PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668(Pharmacia) C225 (lmClone) ZD4190 rhu-Mab (Genentech) (AstraZeneca)ZD6474 MDX-H210 (Medarex) (AstraZeneca) vatalanib (Novartis) 2C4(Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 ABX-EGF (Abgenix)(GlaxoSmithKline) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumabTyrphostins (Genentech) OSI-774 (Tarceva ™) Gefitinib (Iressa) CI-1033(Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3(York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B-Rafenzyme inhibitor, Daiichi Sankyo) Met-MAb (Roche) trametinib (GSK)Additional SR-27897 (CCK A ceflatonin (apoptosis Agents inhibitor,promotor, Sanofi-Synthelabo) ChemGenex) tocladesine (cyclic BCX-1777(PNP inhibitor, AMP agonist, BioCryst) Ribapharm) alvocidib (CDKranpirnase (ribonuclease inhibitor, Aventis) stimulant, Alfacell) CV-247(COX-2 galarubicin (RNA inhibitor, Ivy synthesis inhibitor, Medical)Dong-A) P54 (COX-2 inhibitor, tirapazamine (reducing Phytopharm) agent,SRI International) CapCell ™ (CYP450 N-acetylcysteine (reducingstimulant, agent, Zambon) Bavarian Nordic) GCS-100 (gal3 R-flurbiprofen(NF- antagonist, kappaB inhibitor, GlycoGenesys) Encore) G17DT immunogen3CPA (NF-kappaB (gastrin inhibitor, inhibitor, Active Aphton) Biotech)efaproxiral seocalcitol (vitamin D (oxygenator, Allos receptor agonist,Therapeutics) Leo) PI-88 (heparanase 131-I-TM-601 (DNA inhibitor,Progen) antagonist, TransMolecular) tesmilifene (histamine eflornithine(ODC antagonist, inhibitor, ILEX YM BioSciences) Oncology) histamine(histamine minodronic acid H2 receptor (osteoclast inhibitor, agonist,Maxim) Yamanouchi) tiazofurin (IMPDH indisulam (p53 stimulant,inhibitor, Eisai) Ribapharm) cilengitide (integrin aplidine (PPTinhibitor, antagonist, PharmaMar) Merck KGaA) SR-31747 (IL-1 gemtuzumab(CD33 antagonist, antibody, Wyeth Sanofi-Synthelabo) Ayerst) CCI-779(mTOR PG2 (hematopoiesis kinase inhibitor, enhancer, Wyeth)Pharmagenesis) exisulind (PDE V lmmunol ™ (triclosan oral inhibitor,Cell rinse, Endo) Pathways) CP-461 (PDE V triacetyluridine (uridineinhibitor, Cell prodrug, Wellstat) Pathways) AG-2037 (GART SN-4071(sarcoma agent, inhibitor, Pfizer) Signature BioScience) WX-UK11TransMID-107 ™ (plasminogen (immunotoxin, KS activator inhibitor,Biomedix) Wilex) PBI-1402 (PMN PCK-3145 (apoptosis stimulant, promotor,Procyon) ProMetic LifeSciences) bortezomib doranidazole (apoptosis(proteasome promotor, Pola) inhibitor, Millennium) SRL-172 (T cellCHS-828 (cytotoxic agent, stimulant, SR Leo) Pharma) TLK-286(glutathione trans-retinoic acid S transferase (differentiator, NIH)inhibitor, Telik) PT-100 (growth factor MX6 (apoptosis promotor,agonist, Point MAXIA) Therapeutics) midostaurin (PKC apomine (apoptosisinhibitor, Novartis) promotor, ILEX Oncology) bryostatin-1 (PKC urocidin(apoptosis stimulant, GPC promotor, Bioniche) Biotech) CDA-II (apoptosisRo-31-7453 (apoptosis promotor, Everlife) promotor, La Roche) SDX-101(apoptosis brostallicin (apoptosis promotor, promotor, Salmedix)Pharmacia) rituximab (CD20 β-lapachone antibody, Genentech carmustinegelonin Mitoxantrone cafestol Bleomycin kahweol Absinthin caffeic acidChrysophanic acid Tyrphostin AG Cesium oxides PD-1 inhibitors BRAFinhibitors, CTLA-4 inhibitors PDL1 inhibitors sorafenib MEK inhibitorsBRAF inhibitors bevacizumab angiogenesis inhibitors dabrafenib

An antibody-drug conjugate described herein can be administered with anadditional therapeutic agent concurrently or sequentially (before and/orafter). The antibody-drug conjugate and the additional therapeutic agentcan be administered in the same or different compositions, and by thesame or different routes of administration. A first therapy (which is anantibody-drug conjugate described herein, or the additional therapeuticagent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,or 12 weeks after) the administration of the second therapy (theantibody-drug conjugate described herein, or the additional therapeuticagent) to a subject with cancer. In certain embodiments, an additionaltherapeutic agent administered to a subject in combination with anantibody-drug conjugate is administered in the same composition(pharmaceutical composition). In other embodiments, an additionaltherapeutic agent administered in combination with an antibody-drugconjugate is administered to a subject in a different composition thanthe antibody-drug conjugate (e.g., two or more pharmaceuticalcompositions are used).

5.8. Kits

Also provided herein are kits comprising one or more antibody-drugconjugates described herein. In a specific embodiment, provided hereinis a pharmaceutical pack or kit comprising one or more containers filledwith one or more of the ingredients of the pharmaceutical compositionsdescribed herein, such as one or more antibody-drug conjugates describedherein. In some embodiments, the kits contain a pharmaceuticalcomposition described herein and a prophylactic or therapeutic agent.

Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, a dosage form, and/orinstructions for use thereof. In certain embodiments, the instructionsincluded with the kit provide guidance with respect to the dosageamounts and/or dosing regimens for administration of the pharmaceuticalcomposition(s).

Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, packets, sachets, tubes, inhalers,pumps, bags, vials, containers, syringes and any packaging materialsuitable for a selected pharmaceutical composition and intended mode ofadministration and treatment.

Kits provided herein can further include devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, needle-less injectors, drip bags, patchesand inhalers.

Kits provided herein can further include pharmaceutically acceptablevehicles that can be used to administer the ingredients. For example, ifan ingredient is provided in a solid form that must be reconstituted forparenteral administration, the kit can comprise a sealed container of asuitable vehicle in which the ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration or can be reconstituted as a suspension for oraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: aqueous vehicles including, but notlimited to, Water for Injection USP, Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,and Lactated Ringer's Injection; water-miscible vehicles including, butnot limited to, ethyl alcohol, polyethylene glycol, and polypropyleneglycol; and non-aqueous vehicles including, but not limited to, cornoil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate.

The following examples are offered by way of illustration and not by wayof limitation.

6. EXAMPLES 6.1. Example 1: Generation of High Affinity MERTK-BindingMonoclonal Antibodies

This example describes monoclonal antibodies that inhibit endothelialrecruitment by metastatic breast cancer cells by binding to MERTK andactivating its phosphorylation. The peptide antigen used to immunizemice in order to generate the antibodies described in this example waspurchased from R&D systems (Recombinant Human Mer Fc Chimera,#CF891-MR-100). This is a chimeric peptide comprised of a large portionof the extracellular domain of MERTK (Arg26 to Ala499; SEQ ID NO: 58)fused to a portion of the Fc region of the human IgG1 protein(Pro100-Lys330) with a small linking peptide in between (IEGRMD). SeeFIG. 1A for a schematic of the chimeric peptide. After mice wereimmunized with the chimeric peptide, hybridoma libraries were generatedby fusion of B cells isolated from the immunized mice to myeloma celllines. Supernatant from these hybridomas were then isolated in order toidentify those hybridoma cells generating antibodies that bind MERTK,using antibody capture competitive ELISA assays (FIG. 2 ), as describedin Section 6.1.1, infra. Once identified, hybridomas generatingantibodies with high affinity to MERTK were screened in order toidentify those that generated antibodies capable of inhibiting MERTKfrom binding Gas-6, using antibody capture competitive ELISA assays(FIG. 2 ), as described in Section 6.1.2, infra. To isolate singleclones (monoclonal) hybridoma cells, separation and screening wasperformed on the hybridoma library. 960 single hybridoma clones(monoclonal) were screened from this library to identify those thatgenerated monoclonal antibodies that bound to MERTK with high affinityto neutralize Gas-6 binding.

6.1.1. ELISA Capture Assay for MERTK Binding:

In order to identify monoclonal antibodies that bind to MERTK, an ELISAantibody capture assay was used to screen secreted antibodies generatedfrom each hybridoma clone generated from mice immunized with recombinantMERTK protein. First, polystyrene plates were coated with coatingantibody (Goat anti Mouse IgG (Fc); Lot #: Jackson 98959; antibodyconcentration of 10 ug/mL). Then, supernatant containing primaryantibody from each monoclonal hybridoma clone was added to each well inthe plate. Next, tagged antigen (recombinant human Mer-Fc; RnD systems#891-MR; Lot #: CXK0211051; concentration 300 ng/mL) was added to eachwell. Then, detection antibody (horse radish peroxidase [HRP] conjugatedGoat anti-Human IgG (Fc); Lot #: Jackson 86954; dilution: 1/5000) wasadded to each well. This was followed by addition of HRP substrate andsubsequent addition of stop solution to complete the reaction. Thesignal (O.D.) from each well was read using a spectrophotometer. Of all960 hybridoma clones tested, twenty demonstrated O.D. values>1,indicating significant binding affinity to human MERTK. An O.D. readingof 1 indicates at least 4 times the signal that was observed with thenegative control (empty media without antibody), which had an O.D. valueof 0.22. These twenty clones were named arbitrarily M1 to M20 and arelisted in FIG. 2 . The second column (with the heading “Mer”) in FIG. 2indicates the O.D. reading (indicative of binding affinity) for eachclone.

6.1.2. Competitive Gas-6 Binding ELISA Assay

In order to then identify antibodies that inhibit Gas-6 binding toMERTK, a Gas-6 competitive binding ELISA assay was performed on thetwenty MERTK-binding antibodies (M1-M20). First, polystyrene plates werecoated with recombinant human GAS-6 (RnD systems rhGas6 #885-GS-050;concentration 7 ug/mL). Next, supernatant containing primary antibodyfrom each monoclonal hybridoma clone was added to each well in theplate. Simultaneously, recombinant human MERTK (Mer-Fc RnD systems#891-MR; Lot #: CXK0211051; concentration 300 ng/mL) was added to eachwell. Then, detection antibody (horse radish peroxidase [HRP] conjugatedGoat anti-Human IgG (Fc); Lot #: Jackson 86954; dilution: 1/5000) wasadded to each well. This was followed by the addition of HRP substrateand stop solution to complete the reaction. The signal (O.D.) wasmeasured from each plate using a spectrophotometer. Of all the hybridomaclones tested in this assay, 11 demonstrated O.D. values<2.4, whichindicated they inhibited Gas-6 binding to MERTK. An O.D. reading of 2.4indicates inhibition of Gas-6 binding to MERTK, as the signal that wasobserved with the negative control (empty media without antibody) was2.43. These twenty clones are listed in FIG. 2 . The third column (withthe heading “(Mer+Clone)”) in FIG. 2 indicates the O.D. reading(indicative of amount of inhibition of Gas-6 binding) for each clone.

The two monoclonal antibodies M6 and M19 were selected for furtherstudy, including whether they could inhibit endothelial recruitment bymetastatic cells using trans-well endothelial migration assays.

6.2. Example 2: M19 and M6 Antibodies are Capable of InhibitingEndothelial Cell Recruitment by Triple Negative Breast Cancer Cells InVitro

To identify monoclonal antibodies that could inhibit endothelialrecruitment by activation of MERTK, the high-affinity Gas-6-competingmonoclonal antibodies generated in the screen described in Example 1,supra, were tested in an in vitro endothelial recruitment assay usingtranswells. Metastatic MDA-MB-231 human breast cancer cells were placedin the bottom of a Boyden chamber, where their ability to recruit HUVECSthrough a porous trans-well insert could be assayed. MERTK bindingantibodies from our screen (including both high and low affinityantibodies) were added to the transwells individually in physiologicconcentrations. Of all antibodies tested, M19 and M6 were most able tosignificantly inhibit the recruitment (migrated cells/field) of HUVECcells (50% reduction in migrated cells) versus the negative controlantibody IgG (FIG. 3 ). This demonstrates the ability of the monoclonalantibodies M19 and M6 to inhibit human endothelial cell recruitment byhuman metastatic cancer cells (FIG. 3 ).

6.3. Example 3: M19 and M6 Antibodies Activate MERTK Phosphorylation onEndothelial Cells

To confirm that M19 and M6 are indeed MERTK activating antibodies,western blot analysis was performed to quantify the levels ofphosphorylated (activated) MERTK expressed by endothelial cells (HUVECs)in the presence or absence of M19 or M6 antibody treatment. (FIG. 4 ,FIG. 5 ).

To perform Western blot analysis, HUVEC cells (or LM2 breast cancercells) were grown to 80% confluence in EGM-2 media (Lonza, cat #CC-3162)containing 10% FBS. After a 16 hour incubation under the givenconditions the cells were washed in PBS and lysed in 1 mL RIPA buffer(G-Biosceince, Cat #786-490) containing protease and phosphataseinhibitors (Roche, Cat #11 836 170 001 and 04 906 845 001,respectively). Proteins from the cell lysates were then separated on aSDS-PAGE and transferred to a PVDF membrane. For detection of proteinsthe following primary antibodies were used: Phosphorylated-MERTK(FabGennix, cat #PMKT-140AP), MERTK (Abcam, cat #ab52968),Phosphorylated-Akt (Santa Cruz Biotechnology, cat #sc-7985R), and Akt(Santa Cruz Biotechnology, cat #sc-1618). The primary antibodies weredetected using the appropriate HRP conjugated secondary antibodies (LifeTechnologies).

Cultured cells treated with the specified antibodies resulted in anincrease in activation of MERTK on endothelial cells, in a dosedependent manner during both chronic (16 hour) and acute (30 minute)antibody treatment (FIG. 4 , FIG. 5 ). Interestingly, M19 antibodytreatment did not increase activation of MERTK on LM2 breast cancercells (FIG. 6 ).

6.4. Example 4: M19 Binds with High Affinity to Both Human and MouseMERTK

This example demonstrates that M19 antibody binds with high affinity(K_(D) of ˜0.3 nM) to both human and murine MERTK. To characterize thebinding affinity of M19 antibody against recombinant human and mouseMERTK, biomolecular interaction analysis was performed using biolayerinterferometry (FIG. 7 and FIG. 8 ). Binding was observed between M19and both mouse and human MERTK. The overall global fit calculatedbinding affinity (K_(D)) for M19 binding to human and mouse MERTK weresimilar (K_(D)=˜0.3 nM) (FIG. 7 , FIG. 8 ).

6.5. Example 5: Therapeutic Administration of M19 Inhibits TumorProgression of Human Breast Cancer In Vivo

This example demonstrates that the MERTK activating antibody M19 iscapable of inhibiting tumor progression and tumor metastasis in vivo ina mouse model of human breast cancer. To test whether monoclonalantibody M19 was able to reduce tumor burden and inhibit tumorprogression in vivo, 2000 MDA-MB-231 or 5000 Lm1a1 cells were mixed in a1:1 ratio with growth factor reduced matrigel and injected bilaterallyin the mammary fat pads of NOD-SCID mice. Intraperitoneal injections of250 μg of either M19 antibody or control IgG antibody was injected afterthe surgery and subsequently twice a week. Tumor size of palpable tumorswas measured weekly using a caliber. Treatment with M19 antibody diddemonstrate a significant therapeutic inhibition of both primary tumorgrowth (FIG. 9A) and metastasis (FIG. 9B) of triple-negative breastcancer in vivo.

6.6. Example 6: M19 Inhibits Tumor Angiogenesis In Vivo

To investigate whether therapeutic treatment with M19 antibody resultsin an inhibition of angiogenesis in vivo, blood vessel density withintumors of M19 treated and untreated mice were quantified. 2000MDA-MB-231 were injected bilaterally into the mammary fad pad ofNOD-SCID mice. After 58 days of treatment with 250 μg of either M19antibody or control IgG antibody the tumors were excised and bloodvessel density was quantified (FIG. 10 ). Indeed, therapeutic treatmentwith M19 antibody did result in a significant inhibition (>50%) ofangiogenesis in vivo (FIG. 10 ).

6.7. Example 7: M6 Binds with High Affinity to Both Human MERTK

This example demonstrates that M6 antibody binds with high affinity(K_(D) of ˜4.6 picomolar) to recombinant human MERTK. To characterizethe binding affinity of M6 antibody, biomolecular interation analysiswas performed using biolayer interferometry (FIG. 11 ). Binding wasobserved between M6 and human MERTK. The overall global fit calculatedbinding affinity (K_(D)) for M6 binding to human MERTK was ˜4.6picomolar (FIG. 11 ).

6.8. Example 8: Therapeutic Administration of M6 Inhibits TumorProgression of Human Breast Cancer In Vivo

This example demonstrates that the MERTK activating antibody M6 iscapable of inhibiting tumor progression in vivo in a mouse model ofhuman breast cancer. To test whether monoclonal antibody M6 was able toreduce tumor burden and inhibit tumor progression in vivo, 2000MDA-MB-231 breast cancer cells were mixed in a 1:1 ratio with growthfactor reduced matrigel (BD Biosciences) and injected bilaterally in themammary fat pads of NOD-SCID mice. Intraperitoneal injections of 250 μgof either M6 antibody or control IgG antibody was injected after thesurgery and subsequently twice a week. Tumor size of palpable tumors wasmeasured weekly using a caliber. Indeed, treatment with M6 antibodytwice weekly resulted in a significant reduction in tumor size (FIG. 12).

6.9. Example 9: Antibody Sequences for M6 and M19

To further characterize M19 and M6, the heavy chain and light chainvariable regions of these antibodies were sequenced. The amino acidsequences of both the heavy chain and light chain variable regions of M6are presented in Table 5. The nucleic acid sequences of both the heavychain and light chain variable regions of M6 are presented in Table 7.The amino acid sequences of the CDRs of M6 are presented in Table 1 (forVH) and Table 2 (for VL).

The amino acid sequences of both the heavy chain and light chainvariable regions of M19 are presented in Table 6. The nucleic acidsequences of both the heavy chain and light chain variable regions of M6is presented in Table 8. The amino acid sequences of the CDRs of M19 arepresented in

Table 3 (for VH) and Table 4 (for VL).

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

6.10. Example 10: Treatment of Metastatic Triple-Negative Breast Cancerwith Anti-MERTK Agonistic Antibody-Drug Conjugates

The monoclonal antibody M6 described in Example 1 (Section 6.1) ishumanized and conjugated to the cytotoxic agent monomethyl auristatin F(MMAF) via maleimidocaproic acid (mc) (i.e., the linker). The anti-MERTKantibody-drug conjugate is purified and administered intravenously to apatient who presents with metastatic triple-negative breast cancer. Thepatient is monitored before, during, and after the antibody-drugconjugate administration for response by clinical assessments.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

What is claimed:
 1. An antibody-drug conjugate comprising: (a) anantibody moiety that is an antibody or antigen-binding fragment thereofthat specifically binds to human MERTK; (b) one or more drug moieties,each drug moiety being a cytotoxic agent; and (c) optionally a linker;wherein the cytotoxic agent is conjugated directly to the antibodymoiety or is conjugated to the antibody moiety via the linker; whereinsaid antibody or antigen-binding fragment comprises a heavy chainvariable region (VH) and a light chain variable region (VL), wherein (i)the VH comprises a CDR 1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 6, and aCDR3 of SEQ ID NO: 11; and the VL comprises a CDR1 of SEQ ID NO: 15, aCDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22; or (ii) the VHcomprises a CDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 7, and a CDR3 ofSEQ ID NO: 12; and the VL comprises a CDR1 of SEQ ID NO: 16, a CDR2 ofSEQ ID NO: 20, and a CDR3 of SEQ ID NO: 23; or (iii) the VH comprises aCDR1 of SEQ ID NO: 3, a CDR2 of SEQ ID NO: 8, and a CDR3 of SEQ ID NO:11; and the VL comprises a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO:19 and a CDR3 of SEQ ID NO: 22; or (iv) the VH comprises a CDR1 of SEQID NO: 4, a CDR2 of SEQ ID NO: 9, and a CDR3 of SEQ ID NO: 13; and theVL comprises a CDR1 of SEQ ID NO: 17, a CDR2 of SEQ ID NO: 21, and aCDR3 of SEQ ID NO: 24; or (v) the VH comprises a CDR1 of SEQ ID NO: 5, aCDR2 of SEQ ID NO: 10, and a CDR3 of SEQ ID NO: 14; and the VL comprisesCDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO:22; or (vi) the VH comprises a CDR1 of SEQ ID NO: 3, a CDR2 of SEQ IDNO: 6, and a CDR3 of SEQ ID NO: 11; and the VL comprises a CDR1 of SEQID NO: 15, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 22; or (vii)the VH comprises a CDR1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO: 30, and aCDR3 of SEQ ID NO: 35; and the VL comprises a CDR1 of SEQ ID NO: 39, aCDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO:46; (viii) the VHcomprises a CDR1 of SEQ ID NO: 26, a CDR2 of SEQ ID NO: 31, and a CDR3of SEQ ID NO: 36; and the VL comprises a CDR1 of SEQ ID NO: 40, a CDR2of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 47; or (ix) the VH comprisesa CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 32, and a CDR3 of SEQ IDNO: 35; and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ IDNO: 43, and a CDR3 of SEQ ID NO: 46; or (x) the VH comprises a CDR1 ofSEQ ID NO: 28, a CDR2 of SEQ ID NO: 33, and a CDR3 of SEQ ID NO: 37; andthe VL comprises a CDR1 of SEQ ID NO: 41, a CDR2 of SEQ ID NO: 45, and aCDR3 of SEQ ID NO: 48; or (xi) the VH comprises a CDR1 of SEQ ID NO: 29,a CDR2 of SEQ ID NO: 34, and a CDR3 of SEQ ID NO: 38; and the VLcomprises a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ ID NO: 44, and a CDR3of SEQ ID NO: 46; or (xii) the VH comprises a CDR1 of SEQ ID NO: 27, aCDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35; and the VL comprisesa CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ IDNO:
 46. 2. The antibody-drug conjugate of claim 1, wherein (i) the VHcomprises a CDR 1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 6, and a CDR3 ofSEQ ID NO: 11; and the VL comprises a CDR1 of SEQ ID NO: 15, a CDR2 ofSEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22; or (ii) the VH comprises aCDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO:12; and the VL comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO:20, and a CDR3 of SEQ ID NO: 23; or (iii) the VH comprises a CDR1 of SEQID NO: 3, a CDR2 of SEQ ID NO: 8, and a CDR3 of SEQ ID NO: 11; and theVL comprises a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 19 and a CDR3of SEQ ID NO: 22; or (iv) the VH comprises a CDR1 of SEQ ID NO: 4, aCDR2 of SEQ ID NO: 9, and a CDR3 of SEQ ID NO: 13; and the VL comprisesa CDR1 of SEQ ID NO: 17, a CDR2 of SEQ ID NO: 21, and a CDR3 of SEQ IDNO: 24; or (v) the VH comprises a CDR1 of SEQ ID NO: 5, a CDR2 of SEQ IDNO: 10, and a CDR3 of SEQ ID NO: 14; and the VL comprises CDR1 of SEQ IDNO: 18, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 22; or (vi)the VH comprises a CDR1 of SEQ ID NO: 3, a CDR2 of SEQ ID NO: 6, and aCDR3 of SEQ ID NO: 11; and the VL comprises a CDR1 of SEQ ID NO: 15, aCDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO:
 22. 3. The antibody-drugconjugate of claim 2, wherein the antibody is an immunoglobulin.
 4. Theantibody-drug conjugate of claim 1, wherein the antibody orantigen-binding fragment is humanized.
 5. The antibody-drug conjugate ofclaim 2, wherein the antibody or antigen-binding fragment comprises ahuman-derived constant region.
 6. The antibody-drug conjugate of claim3, wherein the antibody is a humanized immunoglobulin.
 7. Theantibody-drug conjugate of claim 1, wherein the antibody is a bispecificantibody.
 8. An antibody-drug conjugate comprising: (I) an antibodymoiety that is an immunoglobulin that specifically binds to human MERTK,said immunoglobulin comprising: (A) (i) a heavy chain variable regionthat comprises the amino acid sequence of SEQ ID NO: 51, and (ii) alight chain variable region that comprises the amino acid sequence ofSEQ ID NO: 52; or (B) (i) a heavy chain variable region that comprisesthe amino acid sequence of SEQ ID NO: 49, and (ii) a light chainvariable region that comprises the amino acid sequence of SEQ ID NO: 50;(II) one or more drug moieties, each drug moiety being a cytotoxicagent; and (III) optionally a linker; wherein the cytotoxic agent isconjugated directly to the antibody moiety or is conjugated to theantibody moiety via the linker.
 9. An antibody-drug conjugatecomprising: (I) an antibody moiety that is an antibody that competes forbinding to MERTK with a reference antibody selected from the groupconsisting of: (a) a first immunoglobulin comprising (i) a heavy chainvariable region that comprises the amino acid sequence of SEQ ID NO: 51,and (ii) a light chain variable region that comprises the amino acidsequence of SEQ ID NO: 52; and (b) a second immunoglobulin comprising(i) a heavy chain variable region that comprises the amino acid sequenceof SEQ ID NO: 49, and (ii) a light chain variable region that comprisesthe amino acid sequence of SEQ ID NO: 50; (II) one or more drugmoieties, each drug moiety being a cytotoxic agent; and (III) optionallya linker; wherein the cytotoxic agent is conjugated directly to theantibody moiety or is conjugated to the antibody moiety via the linker.10. The antibody-drug conjugate of claim 1, which has a molar ratio ofthe antibody moiety to the drug moiety that is between 1:1 and 1:12. 11.The antibody-drug conjugate of claim 1, wherein the antibody-drugconjugate comprises the linker, and the linker is a cleavable linker.12. The antibody-drug conjugate of claim 1, wherein the antibody-drugconjugate comprises the linker, and the linker is a non-cleavablelinker.
 13. The antibody-drug conjugate of claim 1, wherein thecytotoxic agent is an auristatin, a maytansinoid, apyrrolobenzodiazepine, an indolinobenzodiazepine, a calicheamicin, acamptothecin analogue, a duocarmycin, a tubulin inhibitor, a tubulysinor tubulysin analogue, amberstatin269, doxorubicin, an antibiotic, ananthracycline, a microtubule inhibitor, a spliceostatin, or athailanstatin.
 14. The antibody-drug conjugate of claim 13, wherein thecytotoxic agent is monomethyl auristatin E (MMAE) or monomethylauristatin F (MMAF).
 15. The antibody-drug conjugate of claim 13,wherein the cytotoxic agent is SN38.
 16. A method of producing theantibody-drug conjugate of any of claim 1, wherein said antibody-drugconjugate comprises said linker, said method comprising the followingsteps in the order stated: (a) conjugating the linker directly to thecytotoxic agent to produce a linker-cytotoxic agent moiety; (b)conjugating the linker of the linker-cytotoxic agent moiety directly tothe antibody moiety to produce the antibody-drug conjugate; and (c)purifying the antibody-drug conjugate.
 17. A pharmaceutical compositioncomprising a therapeutically effective amount of the antibody-drugconjugate of claim 1; and a pharmaceutically acceptable carrier.
 18. Amethod of treating cancer in a subject in need thereof, comprisingadministering to said subject the pharmaceutical composition of claim17.
 19. The method of claim 18, wherein said cancer is a cancer of thelung, breast, bone, ovary, stomach, pancreas, larynx, esophagus, testes,liver, parotid, biliary tract, colon, rectum, cervix, uterus,endometrium, kidney, bladder, prostate or thyroid, or said cancer is asarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma,neuroblastoma or Kaposi's sarcoma.
 20. The method of claim 19, whereinsaid cancer is breast cancer.
 21. The method of claim 20, wherein saidcancer is triple-negative breast cancer.
 22. The method of claim 18,wherein said subject is a human.
 23. The antibody-drug conjugate ofclaim 1, wherein (i) the VH comprises a CDR1 of SEQ ID NO: 25, a CDR2 ofSEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35; and the VL comprises a CDR1of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO:46;(ii) the VH comprises a CDR1 of SEQ ID NO: 26, a CDR2 of SEQ ID NO: 31,and a CDR3 of SEQ ID NO: 36; and the VL comprises a CDR1 of SEQ ID NO:40, a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 47; or (iii) theVH comprises a CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 32, and aCDR3 of SEQ ID NO: 35; and the VL comprises a CDR1 of SEQ ID NO: 39, aCDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46; or (iv) the VHcomprises a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 33, and a CDR3of SEQ ID NO: 37; and the VL comprises a CDR1 of SEQ ID NO: 41, a CDR2of SEQ ID NO: 45, and a CDR3 of SEQ ID NO: 48; or (v) the VH comprises aCDR1 of SEQ ID NO: 29, a CDR2 of SEQ ID NO: 34, and a CDR3 of SEQ ID NO:38; and the VL comprises a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ ID NO:44, and a CDR3 of SEQ ID NO: 46; or (vi) the VH comprises a CDR1 of SEQID NO: 27, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35; and theVL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and aCDR3 of SEQ ID NO:
 46. 24. The antibody-drug conjugate of claim 23,wherein the antibody is an immunoglobulin.
 25. The antibody-drugconjugate of claim 24, wherein the antibody is a humanizedimmunoglobulin.
 26. The antibody-drug conjugate of claim 23, wherein theantibody or antigen-binding fragment comprises a human-derived constantregion.
 27. The antibody-drug conjugate of claim 8, wherein theimmunoglobulin comprises (i) a heavy chain variable region thatcomprises the amino acid sequence of SEQ ID NO: 49, and (ii) a lightchain variable region that comprises the amino acid sequence of SEQ IDNO:
 50. 28. A pharmaceutical composition comprising a therapeuticallyeffective amount of the antibody-drug conjugate of claim 2; and apharmaceutically acceptable carrier.
 29. A pharmaceutical compositioncomprising a therapeutically effective amount of the antibody-drugconjugate of claim 3; and a pharmaceutically acceptable carrier.
 30. Apharmaceutical composition comprising a therapeutically effective amountof the antibody-drug conjugate of claim 5; and a pharmaceuticallyacceptable carrier.
 31. A pharmaceutical composition comprising atherapeutically effective amount of the antibody-drug conjugate of claim8; and a pharmaceutically acceptable carrier.
 32. A pharmaceuticalcomposition comprising a therapeutically effective amount of theantibody-drug conjugate of claim 23; and a pharmaceutically acceptablecarrier.
 33. A pharmaceutical composition comprising a therapeuticallyeffective amount of the antibody-drug conjugate of claim 25; and apharmaceutically acceptable carrier.
 34. A method of treating cancer ina subject in need thereof, comprising administering to said subject thepharmaceutical composition of claim
 28. 35. A method of treating cancerin a subject in need thereof, comprising administering to said subjectthe pharmaceutical composition of claim
 29. 36. A method of treatingcancer in a subject in need thereof, comprising administering to saidsubject the pharmaceutical composition of claim
 30. 37. A method oftreating cancer in a subject in need thereof, comprising administeringto said subject the pharmaceutical composition of claim
 31. 38. A methodof treating cancer in a subject in need thereof, comprisingadministering to said subject the pharmaceutical composition of claim32.
 39. A method of treating cancer in a subject in need thereof,comprising administering to said subject the pharmaceutical compositionof claim 33.